Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex

ABSTRACT

A method for identifying nucleic acid ligands to target molecules using the SELEX procedure wherein the candidate nucleic acids contain photoreactive groups and nucleic acid ligands identified thereby are claimed. The complexes of increased affinity nucleic acids and target molecules formed in the procedure are crosslinked by irradiation to facilitate separation from unbound nucleic acids. In other methods partitioning of high and low affinity nucleic acids is facilitated by primer extension steps as shown in the figure in which chain termination nucleotides, digestion resistant nucleotides or nucleotides that allow retention of the cDNA product on an affinity matrix are differentially incorporated into the cDNA products of either the high or low affinity nucleic acids and the cDNA products are treated accordingly to amplification, enzymatic or chemical digestion or by contact with an affinity matrix.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/093,293, filed Jun. 8, 1998 U.S. Pat. No. 6,001,577, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX,” which is acontinuation of U.S. patent application Ser. No. 08/612,895 Mar. 8, 1996U.S. Pat. No. 5,763,177, which is a 35 U.S.C. § 371 filing ofPCT/US94/10562 (WO 95/08003), filed Sep. 16, 1994, entitled “SystematicEvolution of Ligands by Exponential Enrichment: Photoselection ofNucleic Acid Ligands and Solution SELEX,” which is acontinuation-in-part of U.S. patent application Ser. No. 08/123,935,filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,”now abandoned, and a continuation-in-part of U.S. patent applicationSer. No. 08/143,564, filed Oct. 25, 1993, entitled “Systematic Evolutionof Ligands by Exponential Enrichment: Solution SELEX,” abandoned infavor of U.S. patent application Ser. No. 08/461,069, now U.S. Pat. No.5,567,588. U.S. patent application Ser. Nos. 08/123,935 and 08/143,564are continuations-in-part of U.S. patent application Ser. No.07/714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” nowU.S. Pat. No. 5,475,096, which is a continuation-in-part of U.S. patentapplication Ser. No. 07/536,428, filed Jun. 11, 1990, entitled“Systematic Evolution of Ligands by Exponential Enrichment,” nowabandoned. U.S. patent application Ser. No. 08/612,895 is also acontinuation-in-part of U.S. patent application Ser. No. 07/931,473,filed Aug. 17, 1992, entitled “Methods for Identifying Nucleic AcidLigands,” now U.S. Pat. No. 5,270,163, which is a divisional of U.S.patent application Ser. No. 07/714,131.

This work was supported by grants from the United States Governmentfunded through the National Institutes of Health. The U.S. Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

This invention relates, in part, to a method for selecting nucleic acidligands which bind and/or photocrosslink to and/or photoinactivate atarget molecule. The target molecule may be a protein, pathogen or toxicsubstance, or any biological effector. The nucleic acid ligands of thepresent invention contain photoreactive or chemically reactive groupsand are useful, inter alia, for the diagnosis and/or treatment ofdiseases or pathological or toxic states.

The underlying method utilized in this invention is termed SELEX, anacronym for Systematic Evolution of Ligands by EXponential enrichment.An improvement of the SELEX method herein described, termed SolutionSELEX, allows more efficient partitioning between oligonucleotideshaving high and low affinity for a target molecule. An improvement ofthe high affinity nucleic acid products of SELEX are useful for anypurpose to which a binding reaction may be put, for example in assaymethods, diagnostic procedures, cell sorting, as inhibitors of targetmolecule function, as therapeutic agents, as probes, as sequesteringagents and the like.

BACKGROUND OF TE INVENTION

The SELEX method (hereinafter termed SELEX), described in U.S. patentapplication Ser. No. 07/536,428, filed Jun. 11, 1990, entitledSystematic Evolution of Ligands By Exponential Enrichment, nowabandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10,1991, entitled Nucleic Acid Ligands, issued as U.S. Pat. No. 5,475,096and U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992,entitled Methods for Identifying Nucleic Acid Ligands, issued as U.S.Pat. No. 5,270,163, all of which are herein specifically incorporated byreference (referred to herein as the SELEX Patent Applications),provides a class of products which are nucleic acid molecules, eachhaving a unique sequence, each of which has the property of bindingspecifically to a desired target compound or molecule. Each nucleic acidmolecule is a specific ligand of a given target compound or molecule.SELEX is based on the unique insight that nucleic acids have sufficientcapacity for forming a variety of two- and three-dimensional structuresand sufficient chemical versatility available within their monomers toact as ligands (form specific binding pairs) with virtually any chemicalcompound, whether monomeric or polymeric. Molecules of any size canserve as targets.

The SELEX method involves selection from a mixture of candidates andstep-wise iterations of structural improvement, using the same generalselection theme, to achieve virtually any desired criterion of bindingaffinity and selectivity. Starting from a mixture of nucleic acids,preferably comprising a segment of randomized sequence, the methodincludes steps of contacting the mixture with the target underconditions favorable for binding, partitioning unbound nucleic acidsfrom those nucleic acids which have bound to target molecules,dissociating the nucleic acid-target pairs, amplifying the nucleic acidsdissociated from the nucleic acid-target pairs to yield aligand-enriched mixture of nucleic acids, then reiterating the steps ofbinding, partitioning, dissociating and amplifying through as manycycles as desired.

While not bound by theory, SELEX is based on the inventors' insight thatwithin a nucleic acid mixture containing a large number of possiblesequences and structures there is a wide range of binding affinities fora given target. A nucleic acid mixture comprising, for example a 20nucleotide randomized segment can have 4²⁰ candidate possibilities.Those which have the higher affinity constants for the target are mostlikely to bind to the target. After partitioning, dissociation andamplification, a second nucleic acid mixture is generated, enriched forthe higher binding affinity candidates. Additional rounds of selectionprogressively favor the best ligands until the resulting nucleic acidmixture is predominantly composed of only one or a few sequences. Thesecan then be cloned, sequenced and individually tested for bindingaffinity as pure ligands.

Cycles of selection, partition and amplification are repeated until adesired goal is achieved. In the most general case,selection/partition/amplification is continued until no significantimprovement in binding strength is achieved on repetition of the cycle.The method may be used to sample as many as about 10¹⁸ different nucleicacid species. The nucleic acids of the test mixture preferably include arandomized sequence portion as well as conserved sequences necessary forefficient amplification. Nucleic acid sequence variants can be producedin a number of ways including synthesis of randomized nucleic acidsequences and size selection from randomly cleaved cellular nucleicacids. The variable sequence portion may contain fully or partiallyrandom sequence; it may also contain subportions of conserved sequenceincorporated with randomized sequence. Sequence variation in testnucleic acids can be introduced or increased by mutagenesis before orduring the selection/partition/amplification iterations.

Photocrosslinking of nucleic acids to proteins has been achieved throughincorporation of photoreactive functional groups in the nucleic acid.Photoreactive groups which have been incorporated into nucleic acids forthe purpose of photocrosslinking the nucleic acid to an associatedprotein include 5-bromouracil, 4-thiouracil, 5-azidouracil, and8-azidoadenine (see FIG. 1).

Bromouracil has been incorporated into both DNA and RNA by substitutionof bromodeoxyuracil (BrdU) and bromouracil (BrU) for thymine and uracil,respectively. BrU-RNA has been prepared with 5-bromouridine triphosphatein place of uracil using T7 RNA polymerase and a DNA template, and bothBrU-RNA and BrdU-DNA have been prepared with 5-bromouracil and5-bromodeoxyuracil phosphoramidites, respectively, in standard nucleicacid synthesis (Talbot et al. (1990) Nucleic Acids Res. 18:3521). Someexamples of the photocrosslinking of BrdU-substituted DNA to associatedproteins are as follows: BrdU-substituted DNA to proteins in intactcells (Weintraub (1973) Cold Spring Harbor Symp. Quant. Biol. 38:247);BrdU-substituted lac operator DNA to lac repressor (Lin and Riggs (1974)Proc. Natl. Acad. Sci. U.S.A. 71:947; Ogata and Gilbert (1977) Proc.Natl. Acad. Sci. U.S.A. 74:4973; Barbier et al. (1984) Biochemistry23:2933; Wick and Matthews (1991) J. Biol. Chem. 266:6106);BrdU-substituted DNA to EcoRI and EcoRV restriction endonucleases(Wolfes et al. (1986) Eur. J. Biochem. 159:267); Escherichia coliBrdU-substituted DNA to cyclic adenosine 3′,5′-monophosphate receptorprotein (Katouzian-Safadi et al. (1991) Photochem. Photobiol. 53:611);BrdU-substituted DNA oligonucleotide of human polyomavirus to proteinsfrom human fetal brain extract (Khalili et al. (1988) EMBO J. 7:1205); ayeast BrdU-substituted DNA oligonucleotide to GCN4, a yeasttranscriptional activator (Blatter et al. (1992) Nature 359:650); and aBrdU-substituted DNA oligonucleotide of Methanosarcina sp CHT155 to thechromosomal protein Mcl (Katouzian-Safadi et al. (1991) Nucleic AcidsRes. 19:4937). Photocrosslinking of BrU-substituted RNA to associatedproteins has also been reported: BrU-substituted yeast precursortRNA^(Phe) to yeast tRNA ligase (Tanner et al. (1988) Biochemistry27:8852) and a BrU-substituted hairpin RNA of the R17 bacteriophagegenome to R17 coat protein (Gott et al. (1991) Biochemistry 30:6290).

4-Thiouracil-substituted RNA has been used to photocrosslink,especially, t-RNA's to various associated proteins (Favre (1990) in:Bioorganic Photochemistry, Volume 1: Photochemistry and the NucleicAcids, H. Morrison (ed.), John Wiley & Sons: New York, pp. 379-425;Tanner et al. (1988) supra). 4-Thiouracil has been incorporated into RNAusing 4-thiouridine triphosphate and T7 RNA polymerase or using nucleicacid synthesis with the appropriate phosphoramidite; it has also beenincorporated directly into RNA by exchange of the amino group ofcytosine for a thiol group with hydrogen sulfide. Yet another method ofsite specific incorporation of photoreactive groups into nucleic acidsinvolves use of 4-thiouridylyl-(3′-5′)-guanosine (Wyatt et al. (1992)Genes & Development 6:2542).

Examples of 5-azidouracil-substituted and 8-azidoadenine-substitutednucleic acid photocrosslinking to associated proteins are also known.Associated proteins that have been crosslinked include terminaldeoxynucleotidyl transferase (Evans et al. (1989) Biochemistry 28:713;°Farrar et al. (1991) Biochemistry 30:3075); Xenopus TFIIIA, a zincfinger protein (Lee et al. (1991) J. Biol. Chem. 266:16478); and E. coliribosomal proteins (Wower et al. (1988) Biochemistry 27:8114).5-Azidouracil and 8-azidoadenine have been incorporated into DNA usingDNA polymerase or terminal transferase. Proteins have also beenphotochemically labelled by exciting 8-azidoadenosine 3′,5′-biphosphatebound to bovine pancreatic ribonuclease A (Wower et al. (1989)Biochemistry 28:1563) and 8-azidoadenosine 5′-triphosphate bound toribulose-bisphosphate carboxylase/oxygenase (Salvucci and Haley (1990)Planta 181:287).

8-Bromo-2′-deoxyadenosine as a potential photoreactive group has beenincorporated into DNA via the phosphoramidite (Liu and Verdine (1992)Tetrahedron Lett. 33:4265). The photochemical reactivity has yet to beinvestigated.

Photocrosslinking of 5-iodouracil-substituted nucleic acids toassociated proteins has not been previously investigated, probablybecause the size of the iodo group has been thought to preclude specificbinding of the nucleic acid to the protein of interest. However,5-iodo-2′-deoxyuracil and 5-iodo-2′-deoxyuridine triphosphate have beenshown to undergo photocoupling to thymidine kinase from E. coli (Chenand Prusoff (1977) Biochemistry 16:3310).

Mechanistic studies of the photochemical reactivity of the 5-bromouracilchromophore have been reported including studies with regard tophotocrosslinking. Most importantly, BrU shows wavelength dependentphotochemistry. Irradiation in the region of 310 nm populates an n,π*singlet state which decays to ground state and intersystem crosses tothe lowest energy triplet state (Dietz et al. (1987) J. Am. Chem. Soc.109:1793), most likely the π,π* triplet (Rothman and Kearns (1967)Photochem. Photobiol. 6:775). The triplet state reacts withelectron-rich amino acid residues via initial electron transfer followedby covalent bond formation. Photocrosslinking of triplet 5-bromouracilto the electron rich aromatic amino acid residues tyrosine, tryptophanand histidine (Ito et al. (1980) J. Am. Chem. Soc. 102:7535; Dietz andKoch (1987) Photochem. Photobiol. 46:971), and the disulfide bearingamino acid, cystine (Dietz and Koch (1989) Photochem. Photobiol.49:121), has been demonstrated in model studies. Even the peptidelinkage is a potential functional group for photocrosslinking to tripletBrU (Dietz et al. (1987) supra). wavelengths somewhat shorter than 308nm populate both the n,π* and π,π* singlet states. The π,π* singletundergoes carbon-bromine bond homolysis as well as intersystem crossingto the triplet manifold (Dietz et al. (1987) supra); intersystemcrossing may occur in part via internal conversion to the n,π* singletstate. Carbon-bromine bond homolysis likely leads to nucleic acid strandbreaks (Hutchinson and Kohnlein (1980) Prog. Subcell. Biol. 7:1; Shetlar(1980) Photochem. Photobiol. Rev. 5:105; Saito and Sugiyama (1990) in:Bioorganic Photochemistry, Volume 1: Photochemistry and the NucleicAcids, H. Morrison, ed., John Wiley and Sons, New York, pp. 317-378).The wavelength dependent photochemistry is outlined in the JablonskiDiagram in FIG. 2 and the model photocrosslinking reactions are shown inFIG. 3.

The location of photocrosslinks from irradiation of some BrU-substitutednucleoprotein complexes have been investigated. In the lacrepressor-BrdU-lac operator complex a crosslink to tyrosine-17 has beenestablished (Allen et al. (1991) J. Biol. Chem. 266:6113). In thearchaebacterial chromosomal protein MC1-BrdU-DNA complex a crosslink totryptophan-74 has been implicated. In yeast BrdU-substituted DNA-GCN4yeast transcriptional activator a crosslink to alanine-238 was reported(Blatter et al. (1992) supra). In this latter example the nucleoproteincomplex was irradiated at 254 nm which populated initially the π,π*singlet state.

The results of some reactivity and mechanistic studies of 5-iodouracil,5-iodo-2′-deoxyuracil, 5-iodo-2′-deoxyuracil-substituted DNA, and5-iodo-2′-deoxycytosine-substituted DNA have been reported. 5-Iodouraciland 5-iodo-2′-deoxyuracil couple at the 5-position to allylsilanes uponirradiation in acetonitrile-water bearing excess silane with emissionfrom a medium pressure mercury lamp filtered through Pyrex glass; themechanism was proposed to proceed through initial carbon-iodine bondhomolysis followed by radical addition to the π-bond of the allylsilane(Saito et al. (1986) J. Org. Chem. 51:5148).

Aerobic and anaerobic photo-deiodination of5-iodo-2′-deoxyuracil-substituted DNA has been studied as a function ofexcitation wavelength; the intrinsic quantum yield drops by a factor of4 with irradiation in the region of 313 nm relative to the quantum yieldwith irradiation in the region of 240 nm. At all wavelengths themechanism is proposed to involve initial carbon-iodine bond homolysis(Rahn and Sellin (1982) Photochem. Photobiol. 35:459). Similarly,carbon-iodine bond homolysis is proposed to occur upon irradiation of5-iodo-2′-deoxycytidine-substituted DNA at 313 nm (Rahn and Stafford(1979) Photochem. Photobiol. 30:449). Strictly monochromatic light wasnot used in any of these studies. Recently, a 5-iodouracil-substitutedduplex DNA was shown to undergo a photochemical single strand break(Sugiyama et al. (1993) J. Am. Chem. Soc. 115:4443).

Also of importance with respect to the present invention is the observeddirect population of the triplet states of 5-bromouracil and5-iodouracil from irradiation of the respective S_(o)→T absorption bandsin the region of 350-400 nm (Rothman and Kearns (1967) supra).

Photophysical studies of the 4-thiouracil chromophore implicate the π,π*triplet state as the reactive state. The intersystem crossing quantumyield is unity or close to unity. Although photocrosslinking within4-thiouracil-substituted nucleoprotein complexes has been observed,amino acid residues reactive with excited 4-thiouracil have not beenestablished (Favre (1990) supra). The addition of the α-amino group oflysine to excited 4-thiouracil at the 6-position has been reported;however, this reaction is not expected to be important inphotocrosslinking within nucleoprotein complexes because the α-aminogroup is involved in a peptide bond (Ito et al. (1980) Photochem.Photobiol. 32:683).

Photocrosslinking of azide-bearing nucleotides or nucleic acids toassociated proteins is thought to proceed via formation of the singletand/or triplet nitrene (Bayley and Knowles (1977) Methods Enzymol.46:69; Czarnecki et al. (1979) Methods Enzymol. 56:642; Hanna et al.(1993) Nucleic Acids Res. 21:2073). Covalent bond formation results frominsertion of the nitrene in an O—H, N—H, S—H or C—H bond. Singletnitrenes preferentially insert in heteroatom-H bonds and tripletnitrenes in C—H bonds. Singlet nitrenes can also rearrange to azirineswhich are prone to nucleophilic addition reactions. If a nucleophilicsite of a protein is adjacent, crosslinking can also occur via thispathway. A potential problem with the use of an azide functional groupresults if it resides ortho to a ring nitrogen; the azide will exist inequilibrium with a tetrazole which is much less photoreactive.

The coat protein-RNA hairpin complex of the R17 bacteriophage is anideal system for the study of nucleic acid-protein photocrosslinkingbecause of the simplicity of the system in vitro. The system is wellcharacterized, consisting of a viral coat protein that binds with highaffinity to an RNA hairpin within the phage genome. In vivo theinteraction of the coat protein with the RNA hairpin plays two rolesduring phage infection: the coat protein acts as a translationalrepressor of replicase synthesis (Eggen and Nathans (1969) J. Mol. Biol.39:293), and the complex serves as a nucleation site for encapsidation(Ling et al. (1970) Virology 40:920; Beckett et al. (1988) J. Mol Biol.204:939). Many variations of the wild-type hairpin sequence also bind tothe coat protein with high affinity (Tuerk & Gold (1990) Science249:505; Gott et al. (1991) Biochemistry 30:6290; Schneider et al.(1992) J. Mol. Biol. 228:862).

The selection of nucleic acid ligands according to the SELEX method maybe accomplished in a variety of ways, such as on the basis of physicalcharacteristics. Selection on the basis of physical characteristics mayinclude physical structure, electrophoretic mobility, solubility, andpartitioning behavior. U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled Method for Selecting Nucleic Acids on theBasis of Structure, now abandoned (See; U.S. Pat. No. 5,707,796) hereinspecifically incorporated by reference, describes the selection ofnucleic acid sequences on the basis of specific electrophoreticbehavior. The SELEX technology may also be used in conjunction withother selection techniques, such as HPLC, column chromatography,chromatographic methods in general, solubility in a particular solvent,or partitioning between two phases.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a method for selectingand identifying nucleic acid ligands from a candidate mixture ofrandomized nucleic acid sequences on the basis of the ability of therandomized nucleic acid sequences to bind and/or photocrosslink to atarget molecule. This embodiment is termed Covalent SELEX generally, andPhotoSELEX specifically when irradiation is required to form covalentlinkage between the nucleic acid ligand and the target.

In one variation of this embodiment, the method comprises preparing acandidate mixture of nucleic acid sequences which contain photoreactivegroups; contacting the candidate mixture with a target molecule whereinnucleic acid sequences having increased affinity to the target moleculebind the target molecule, forming nucleic acid-target moleculecomplexes; irradiating the nucleic acid-target molecule mixture, whereinsome nucleic acids incorporated in nucleic acid-target moleculecomplexes crosslink to the target molecule via the photoreactivefunctional groups; taking advantage of the covalent bond to partitionthe crosslinked nucleic acid-target molecule complexes from free nucleicacids in the candidate mixture; and identifying the nucleic acidsequences that were photocrosslinked to the target molecule. The processcan further include the iterative step of amplifying the nucleic acidsthat photocrosslinked to the target molecule to yield a mixture ofnucleic acids enriched in sequences that are able to photocrosslink tothe target molecule.

In another variation of this embodiment of the present invention,nucleic acid ligands to a target molecule selected through SELEX arefurther selected for their ability to crosslink to the target. Nucleicacid ligands to a target molecule not containing photoreactive groupsare initially identified through the SELEX method. Photoreactive groupsare then incorporated into these selected nucleic acid ligands, and theligands contacted with the target molecule. The nucleic acid-targetmolecule complexes are irradiated and those able to photocrosslink tothe target molecule identified.

In another variation of this embodiment of the present invention,photoreactive groups are incorporated into all possible positions in thenucleic acid sequences of the candidate mixture. For example,5-iodouracil and 5-iodocytosine may be substituted at all uracil andcytosine positions. The first selection round is performed withirradiation of the nucleic acid-target molecule complexes such thatselection occurs for those nucleic acid sequences able to photocrosslinkto the target molecule. Then SELEX is performed with the nucleic acidsequences able to photocrosslink to the target molecule to selectcrosslinking sequences best able to bind the target molecule.

In another variation of this embodiment of the present invention,nucleic acid sequences containing photoreactive groups are selectedthrough SELEX for a number of rounds in the absence of irradiation,resulting in a candidate mixture with a partially enhanced affinity forthe target molecule. PhotoSELEX is then conducted with irradiation toselect ligands able to photocrosslink to the target molecule.

In another variation of this embodiment of the present invention, SELEXis carried out to completion with nucleic acid sequences not containingphotoreactive groups, and nucleic acid ligands to the target moleculeselected. Based on the sequences of the selected ligands, a family ofrelated nucleic acid sequences is generated which contain a singlephotoreactive group at each nucleotide position. PhotoSELEX is performedto select a nucleic acid ligand capable of photocrosslinking. to thetarget molecule.

In a further variation of this embodiment of the present invention, anucleic acid ligand capable of modifying the bioactivity of a targetmolecule through binding and/or crosslinking to a target molecule isselected through SELEX, photoSELEX, or a combination of these methods.

In a further variation of this embodiment of the present invention, anucleic acid ligand to a unique target molecule associated with aspecific disease process is identified. In yet another variation of thisembodiment of the present invention, a nucleic acid ligand to a targetmolecule associated with a disease state is used to treat the disease invivo.

The present invention further encompasses nucleic acid sequencescontaining photoreactive groups. The nucleic acid sequences may containsingle or multiple photoreactive groups. Further, the photoreactivegroups may be the same or different in a single nucleic acid sequence.The photoreactive groups incorporated into the nucleic acids of theinvention include any chemical group capable of forming a crosslink witha target molecule upon irradiation. Although in some cases irradiationmay not be necessary for crosslinking to occur.

The nucleic acids of the present invention include single- anddouble-stranded RNA and single- and double-stranded DNA. The nucleicacids of the present invention may contain modified groups such as2′-amino (2′-NH₂) or 2′-fluoro (2′-F)-modified nucleotides. The nucleicacids of the present invention may further include backbonemodifications.

The present invention further includes the method whereby candidatemixtures containing modified nucleic acids are prepared and utilized inthe SELEX process, and nucleic acid ligands are identified that bind orcrosslink to the target species. In one example of this embodiment, thecandidate mixture is comprised of nucleic acids wherein all uracilresidues are replaced by 5-halogenated uracil residues, and nucleic acidligands are identified that form covalent attachments to the selectedtarget.

An additional embodiment of the present invention, termed solutionSELEX, presents several improved methods for partitioning betweenligands having high and low affinity nucleic acid-target complexes isachieved in solution and without, or prior to, use of a partitioningmatrix. Generally, a central theme of the method of solution SELEX isthat the nucleic acid candidate mixture is treated in solution andresults in preferential amplification during PCR of the highest affinitynucleic acid ligands or catalytic RNAs. The solution SELEX methodachieves partitioning between high and low affinity nucleic acid-targetcomplexes through a number of methods, including (1) Primer extensioninhibition which results in differentiable CDNA products such that thehighest affinity ligands may be selectively amplified during PCR. Primerextension inhibition is achieved with the use of nucleic acidpolymerases, including DNA or RNA polymerases, reverse transcriptase,and Qβ-replicase. (2) Exonuclease hydrolysis inhibition which alsoresults in only the highest affinity ligands amplifying during PCR. Thisis achieved with the use of any 3′→5 double-stranded exonuclease. (3)Linear to circle formation to generate differentiable cDNA moleculesresulting in amplification of only the highest affinity ligands duringPCR.

In one embodiment of the solution SELEX method, synthesis of cDNAscorresponding to low affinity oligonucleotides are preferentiallyblocked and thus rendered non-amplifiable by PCR. In another embodiment,low affinity oligonucleotides are preferentially removed by affinitycolumn chromatography prior to PCR amplification. Alternatively, highaffinity oligonucleotides may be preferentially removed by affinitycolumn chromatography. In yet another embodiment of the SELEX method,cDNAs corresponding to high affinity oligonucleotides are preferentiallyrendered resistant to nuclease enzyme digestion. In a furtherembodiment, cDNAs corresponding to low affinity oligonucleotides arerendered preferentially enzymatically or chemically degradable.

Solution SELEX is an improvement over prior art partitioning schemes.With the method of the present invention, partitioning is achievedwithout inadvertently also selecting ligands that only have affinity forthe partitioning matrix, the speed and accuracy of partitioning isincreased, and the procedure may be readily automated.

The present disclosure provides non-limiting examples which areillustrative and exemplary of the invention. Other partitioning schemesand methods of selecting nucleic acid ligands through binding andphotocrosslinking to a target molecule will be become apparent to oneskilled in the art from the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows structures of photoreactive chromophores which have beenincorporated into nucleic acids.

FIG. 2 shows a Jablonski energy level diagram for the 5-bromouracilchromophore and the reactivity of the various excited states.

FIG. 3 shows the model reactions for photocrosslinking of the5-bromouracil chromophore to amino acid residues such as tyrosine,tryptophan, histidine, and cystine.

FIG. 4 compares UV absorption by thymidine, 5-bromouracil, 5-iodouracil,and L-tryptophan in TMK pH 8.5 buffer (100 mMtris(hydroxymethyl)aminomethane hydrochloride, 10 mM magnesium acetate,and 80 mM potassium chloride). The emission wavelengths of the XeCl andHeCd lasers are also indicated. Of particular importance is absorptionby 5-iodouracil at 325 nm without absorption by tryptophan or thymidine.The molar extinction coefficient for 5-iodouracil at 325 nm is 163L/mol·cm.

FIG. 5 shows structures of photoreactive chromophores which can beincorporated into randomized nucleic acid sequences.

FIG. 6 shows the structures of hairpin RNA sequences RNA-1 (SEQ IDNO:1), RNA-2 (SEQ ID NO:2), and RNA-3 (SEQ ID NO:3) containing5-bromouracil, 5-iodouracil, and uracil, respectively. These arevariants of the wild-type hairpin of the R17 bacteriophage genome whichbind tightly to the R17 coat protein.

FIG. 7 shows binding curves for RNA-1 (SEQ ID NO:1), RNA-2 (SEQ IDNO:2), and RNA-3 (SEQ ID NO:3) to R17 coat protein. The bindingconstants calculated from the binding curves are also given.

FIG. 8 shows the percent of RNA-1 (SEQ ID NO:1) and RNA-2 (SEQ ID NO:2)photocrosslinked to R17 coat protein with monochromatic irradiation at308 nm from a XeCl excimer laser as a function of time.Photocrosslinking of RNA-1 (SEQ ID NO:1) maximized at 40% because ofcompetitive photodamage to the coat protein during the irradiationperiod. Less photodamage to coat protein occurred with RNA-2 (SEQ IDNO:2) because of the shorter irradiation time.

FIG. 9 shows the percent of RNA-2 (SEQ ID NO:2) photocrosslinked to R17coat protein with monochromatic irradiation at 325 nm from a HeCd laseras a function of time. The data are also presented in the originalelectrophoretic gel format. The symbol IU XL marks RNA crosslinked toprotein. A near-quantitative yield of photocrosslinking was obtained.

FIG. 10 shows the percent of RNA-1 (SEQ ID NO:1) and RNA-2 (SEQ ID NO:2)photocrosslinked to R17 coat protein with broad-band irradiation in theregion of 312 nm from a Transilluminator as a function of time. Lessthan quantitative yields of photocrosslinking were obtained because ofphotodamage to the protein and possibly to the RNA sequences.

FIG. 11 shows formation of the same product, Structure 6, fromirradiation at 308 nm of 5-iodouracil and 5-bromouracil in the presenceof excess N-acetyltyrosine N-ethylamide (Structure S).

FIG. 12 pictures photocrosslinking of RNA-7 (SEQ ID NO:4) to R17 coatprotein with 308 nm light followed by enzymatic digestion of most of thecoat protein.

FIG. 13 shows formation of a complementary DNA from the RNA templateafter enzymatic digestion of the coat protein of FIG. 12 (SEQ ID NO:4).

FIG. 14 shows the polyacrylamide gel of Example 8 showing production ofa cDNA from an RNA template bearing modified nucleotides as shown inFIGS. 12 and 13. The modified nucleotides were 5-iodouracil and uracilsubstituted at the 5-position with a small peptide. Based upon modelstudies shown in FIG. 11, the peptide was most likely attached to theuracil via the phenolic ring of a tyrosine residue.

FIG. 15 shows the photocrosslinking of [α-³²P] GTP labelled pool RNA toHIV-1 Rev protein using tRNA competition.

FIG. 16 (SEQ ID NOS:5-55) shows the sequence of the previouslyidentified RNA ligand to HIV-1 Rev protein that is referred to herein as6a (SEQ ID NO:5). Also shown are 52 sequences from round 13 selected forphotocrosslinking to HIV-1 Rev protein.

FIG. 17 (SEQ ID NOS:56-57) shows the consensus for class 1 ligands andclass 2 ligands. Class 1: Consensus secondary structure for class 1 andclass 2 molecules. N₁-N₁′ indicate 1-2 complementary base pairs; N₂-N₂′indicates 1-4 complementary base pairs, D-H′ is an A-U, U-A, or G-C basepair; K-M′ is a G-C or U-A base pair (16). Class 2: Bold sequencesrepresent the highly conserved 10 nucleotides that characterize class 2molecules; base-pairing is with the 5′ fixed end of the molecule.

FIG. 18 (SEQ ID NO:58) shows the sequence and predicted secondarystructure of trunc2 (FIG. 18A). Also shown (FIG. 18B) is a geldemonstrating the specificity of trunc2 photocrosslinking to ARMproteins.

FIG. 19 shows the sequence and predicted secondary structure of trunc24(SEQ ID NO:59) (FIG. 19A). Also shown (FIG. 19B) is a gel demonstratingthe specificity of laser independent crosslinking to ARM proteins.

FIG. 20 shows the trunc24 (SEQ ID NO:59) photo-independent crosslinkingwith HIV-1 Rev in the presence of human nuclear extracts.

FIG. 21 illustrates the cyclical relationship between SELEX steps. Asingle-stranded nucleic acid repertoire of candidate oligonucleotides isgenerated by established procedures on a nucleic acid synthesizer, andis amplified by PCR to generate a population of double-stranded DNAmolecules. Candidate DNA or RNA molecules are generated throughasymmetric PCR or transcription, respectively, purified, and allowed tocomplex with a target molecule. This is followed by partitioning ofbound and unbound nucleic acids, synthesis of cDNA, and PCRamplification to generate double-stranded DNA.

FIG. 22 illustrates one embodiment of the solution SELEX method in whichprimer extension inhibition is used to create differentiable cDNApools—an amplifiable high affinity oligonucleotide cDNA pool and anon-amplifiable low affinity oligonucleotide CDNA pool. In thisembodiment, the first cDNA extension is performed in the presence ofchain terminating nucleotides such as ddG. After removal of the targetmolecule and dideoxynucleotides, the second cDNA extension is conductedin the presence of four dNTPs. Full-length cDNA is preferentiallysynthesized from the high affinity oligonucleotides and therefore, thehigh affinity cDNA pool is amplified in the subsequent PCR step.

FIG. 23 illustrates the cyclic solution SELEX process for the embodimentshown in FIG. 22.

FIG. 24 illustrates one embodiment of the cyclic solution SELEX processwherein partitioning between oligonucleotides having high and lowaffinity to a target molecule is achieved by restriction enzymedigestion. In this embodiment, the first cDNA extension is conductedwith four dNTPs and results in cDNAs corresponding to the low affinityoligonucleotides. The target is then removed and a second cDNA extensionis conducted in the presence of modified nucleotides resistant toenzymatic cleavage. The cDNA pools are then incubated with restrictionenzyme and only the cDNA pool corresponding to high affinityoligonucleotides is amplifiable in the subsequent PCR step.

FIG. 25 illustrates one embodiment of the cyclic solution SELEX processwherein partitioning between oligonucleotides having high and lowaffinity to a target molecule is achieved by affinity chromatography.The first cDNA extension is performed in the presence of a modifiednucleotide such as a biotinylated nucleotide, which allows the cDNA poolcorresponding to the low-affinity oligonucleotide to be retained on anaffinity column.

FIG. 26 illustrates one embodiment of the solution SELEX process whereinpartitioning between oligonucleotides having high and low affinity to atarget molecule is achieved by exonuclease inhibition and results information of a double-stranded nucleic acid population with highaffinity for the target molecule.

FIG. 27 illustrates one embodiment of the solution SELEX process whereincatalytic nucleic acids are selected and isolated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes a variation of the SELEX method forselecting nucleic acid ligands. This application hereby specificallyincorporates by reference the full text including the definitionsprovided in the earlier SELEX patent applications, specifically thoseprovided in U.S. patent application Ser. No. 07/536,428, filed Jun. 11,1990, now abandoned, and 07/714,131, filed Jun. 10, 1991, now U.S. Pat.No. 5,475,096. The method of one embodiment of the present invention,termed covalent SELEX or photoSELEX, identifies and selects nucleic acidligands capable of binding and/or photocrosslinking to target molecules.

This application also presents a method for improved partitioning ofnucleic acid ligands identified through the SELEX method.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: a) to assistin the amplification steps described below; b) to mimic a sequence knownto bind to the target; or c) to enhance the potential of a givenstructural arrangement of the nucleic acids in the candidate mixture.The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with the selected target underconditions favorable for binding between the target and members of thecandidate mixture. Under these circumstances, the interaction betweenthe target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthe nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-10%) is retainedduring partitioning.

4) Those nucleic acids selected during partitioning as having therelatively higher affinity to the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newlyformed candidate mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the nucleic acid mixture to the targetwill generally increase. Taken to its extreme, the SELEX process willyield a candidate mixture containing one or a small number of uniquenucleic acids representing those nucleic acids from the originalcandidate mixture having the highest affinity to the target molecule.

The SELEX Patent Applications describe and elaborate on this process ingreat detail. Included are targets that can be used in the process;methods for the preparation of the initial candidate mixture; methodsfor partitioning nucleic acids within a candidate mixture; and methodsfor amplifying partitioned nucleic acids to generate enriched candidatemixtures. The SELEX Patent Applications also describe ligand solutionsobtained to a number of target species, including both protein targetswherein the protein is and is not a nucleic acid binding protein.

Partitioning means any process whereby ligands bound to target moleculescan be separated from nucleic acids not bound to target molecules. Morebroadly stated, partitioning allows for the separation of all thenucleic acids in a candidate mixture into at least two pools based ontheir relative affinity to the target molecule. Partitioning can beaccomplished by various methods known in the art. Nucleic acid-proteinpairs can be bound to nitrocellulose filters while unbound nucleic acidsare not. Columns which specifically retain nucleic acid-target complexescan be used for partitioning. For example, oligonucleotides able toassociate with a target molecule bound on a column allow use of columnchromatography for separating and isolating the highest affinity nucleicacid ligands. Liquid-liquid partitioning can be used as well asfiltration gel retardation, and density gradient centrifugation.

I. PhotoSELEX.

The present invention encompasses nucleic acid ligands which bind,photocrosslink and/or photoinactivate target molecules. Binding asreferred to herein generally refers to the formation of a covalent bondbetween the ligand and the target, although such binding is notnecessarily irreversible. Certain nucleic acid ligands of the presentinvention contain photoreactive-groups which are capable ofphotocrosslinking to the target molecule upon irradiation with light.Additional nucleic acid ligands of the present invention are capable ofbond formation with the target in the absence of irradiation.

In one embodiment, the present invention encompasses nucleic acidligands which are single- or double-stranded RNA or DNAoligonucleotides. The nucleic acid ligands of the present invention maycontain photoreactive groups capable of crosslinking to the selectedtarget molecule when irradiated with light. Further, the presentinvention encompasses nucleic acid ligands containing any modificationthereof. Reference to a photoreactive group herein may simply refer to arelatively simple modification to a natural nucleic acid residue thatconfers increased reactivity or photoreactivity to the nucleic acidresidue. Such modifications include, but are not limited to,modifications at cytosine exocyclic amines, substitution withhalogenated groups, e.g., 5′-bromo- or 5′-iodo-uracyl, modification atthe 2′-5 position, e.g., 2′-amino (2′-NH₂) and 2′-fluoro (2′- F),backbone modifications, methylations, unusual base-pairing combinationsand the like. For example, the nucleic acid ligands of the presentinvention may include modified nucleotides such as 2′-NH₂-iodouracil,2′-NH₂-iodocytosine, 2′-NH₂-iodoadenine, 2′-NH₂-bromouracil,2′-NH₂-bromocytosine, and 2′-NH₂-bromoadenine.

In one embodiment of the photoSELEX method of the present invention, arandomized set of nucleic acid sequences containing photoreactivegroups, termed the candidate mixture, is mixed with a quantity of thetarget molecule and allowed to establish an equilibrium binding with thetarget molecule. The nucleic acid-target molecule mixture is thenirradiated with light until photocrosslinking is complete as indicatedby polyacrylamide gel electrophoresis. Only some of those nucleic acidsbinding tightly to the target molecules will efficiently crosslink withthe target.

The candidate mixture of the present invention is comprised of nucleicacid sequences with randomized regions including chemically reactive ora photoreactive group or groups. Preferably the reactive groups aremodified nucleic acids. The nucleic acids of the candidate mixturepreferably include a randomized sequence portion as well as conservedsequences necessary for efficient amplification. The variable sequenceportion may contain fully or partially random sequence; it may alsocontain subportions of conserved sequence incorporated within therandomized sequence regions.

Preferably, each oligonucleotide member of the candidate mixturecontains at least one chemically reactive or photoreactive group.Further, each oligonucleotide member of the candidate mixture may bepartially or fully substituted at each position by modified nucleotidescontaining reactive groups. The candidate mixture may also be comprisedof oligonucleotides containing more than one type of reactive group.

The target molecules bound and/or photocrosslinked by the nucleic acidligands of the present invention are commonly proteins and are selectedbased upon their role in disease and/or toxicity. Examples are enzymesfor which an inhibitor is desired or proteins for which detection isdesired. However, the target molecule may be any compound of interestfor which a ligand is desired. A target molecule can be a protein,peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, substrate, metabolite, transition stateanalog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc.,without limitation.

A photoreactive group for the purpose of this application is preferablya modified nucleotide that contains a photochromophore, and that iscapable of photocrosslinking with a target species. Although referred toherein as photoreactive groups, in some cases as described below,irradiation is not necessary for covalent binding to occur between thenucleic acid ligand and the target. Preferentially, the photoreactivegroup will absorb light in a spectrum of the wavelength that is notabsorbed by the target or the non-modified portions of theoligonucleotide. This invention encompasses, but is not limited to,oligonucleotides containing a photoreactive group selected from thefollowing: 5-bromouracil (BrU), 5-iodouracil (IU), 5-bromovinyluracil,5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine,5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine,5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine,8-azidoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine,8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine,8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuridine,8-bromo-2′-deoxyadenine, 5-iodo-2′-deoxyuracil, 5-iodo-2′-deoxycytosine,5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil,7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine,and 7-deaza-7-bromoguanine (FIG. 5). In one embodiment, thephotoreactive groups are 5-bromouracil (BrU) and 5-iodouracil (IU).

The photoreactive groups of the present invention are capable of formingbonds with the target species upon irradiation of an associated nucleicacid-target pair. The associated pair is referred to herein as anucleoprotein complex, and in some cases irradiation is not required forbond formation to occur. The photocrosslink that typically occurs willbe the formation of a covalent bond between the associated nucleic acidand the target. However, a tight ionic interaction between the nucleicacid and target may also occur upon irradiation.

In one embodiment, photocrosslinking occurs due to electromagneticirradiation. Electromagnetic irradiation includes ultraviolet light,visible light, X-ray and gamma ray. 5-Halo substituted deoxyuracils anddeoxycytosines are known to sensitize cells to ionizing radiation(Szybalski (1974) Cancer Chemother. Rep. 58:539).

Crosslinking experiments have shown that a precise juxtaposition ofeither IU or BrU and a tyrosine, tryptophan, or histidine is requiredfor a high yield crosslinking to occur. The present invention takesadvantage of this finding with selection for crosslinking molecules withrandomly incorporated photoreactive groups. In one embodiment of thepresent invention, the photoreactive groups 5-bromouracil (BrU) or5-iodouracil (IU) are incorporated into RNA by T7 polymerasetranscription with the 5-halouridine triphosphate present in place ofuridine triphosphate. Incorporation is achieved by using a mixture of5-halouridine triphosphate and uridine triphosphate or all 5-halouridinetriphosphate. A randomized set of ³²P or ³³P-labeled or unlabeled RNAsequences is obtained from a randomized set of DNA templates,synthesized using standard methodology.

The randomized set of RNA oligonucleotides containing BrU or IU aremixed with a quantity of a target protein. The photoreactive chromophoreis incorporated randomly into RNA as BrU or IU in place of uracil usingstandard methodology. The RNA-target protein mixture is irradiated withnear ultraviolet light in the range of 300 to 325 nm untilphotocrosslinking is complete. Only those photoreactive groups adjacentto a reactive amino acid residue in a nucleoprotein complex form acovalent bond to the protein. Excited BrU or IU, returns to the groundstate unless it is near a reactive functional group such as anoxidizable amino acid residue. Amino acid residues which have beenestablished as being reactive with the lowest triplet state of5-bromouracil include tyrosine, tryptophan, histidine, and cystine (seeFIG. 3). Others of potential reactivity based upon mechanistic studiesare phenylalanine, methionine, cysteine, lysine, and arginine.

Nucleoprotein complexes which do not form crosslinks may be easilydisrupted by adjusting the reaction medium such as by denaturing withheat and/or salt. Nucleic acids covalently bound to the protein may beseparated from free nucleic acids on a nitrocellulose filter or by otherpartitioning methods known to those skilled in the art. Alternatemethods for separating nucleic acids covalently bound to targets fromfree nucleic acids include gel electrophoresis followed byelectroelution, precipitation, differential solubility, andchromatography. To one skilled in the art, the method of choice willdepend at least in part on the target molecule of interest. Thecrosslinked nucleic acids are released from the nitrocellulose filter bydigestion of the protein material with enzymes such as Proteinase K. Atthis point 5-halouracil groups which have photocrosslinked to the targetprotein are bound to a single amino acid or to a short peptide. Theread-through ability of reverse transcriptase is not effected byincorporation of a substituent at the 5-position of uracil becausereverse transcriptase (RT) does not differentiate the 5-position ofuracil from that of thymine. Derivatization of the 5-position has beenused to incorporate groups as large as biotin into RNA molecules. In oneembodiment of the present invention, the target is removed from theselected photocrosslinked nucleic acid by photo or chemicaldissociation.

Complementary nucleic acid copies of the selected RNA sequences areprepared with an appropriate primer. The cDNA is amplified with a DNApolymerase and a second primer. 5-Halo-2′-deoxyuracil is not employed inthe DNA synthesis and amplification steps. The amplified DNAs are thentranscribed into RNA sequences using 5-halouridine triphosphate in placeof uridine triphosphate in the same or different ratio of 5-halouridineto uridine in the candidate mixture.

For the subsequent round of photoSELEX, the partially selected RNAsequences are again allowed to complex with a quantity of the targetprotein. Subsequently, the nucleoprotein complexes are irradiated in theregion of 300-325 nm. RNA sequences which have crosslinked to proteinare again separated from RNA sequences which have not crosslinked. cDNAsare prepared and amplified and a third set of RNA sequences containing5-halouracil are prepared. The cycle is continued until it converges toone or several RNA ligands which bind with high affinity andphotocrosslink to the target protein. Shortening of the irradiation timein later cycles can further enhance the selection. The cDNAs of theselected RNA ligands are amplified, gel purified, and sequenced.Alternatively, the RNA sequences can be sequenced directly. The selectedRNA sequences are then transcribed from the appropriate synthesized DNAtemplate, again using 5-halouridine triphosphate in place of uridinetriphosphate (Example 11).

In another embodiment of the present invention, photoSELEX is performedon oligonucleotide sequences preselected for their ability to bind thetarget molecule (Example 12). SELEX is initially performed witholigonucieotides which do not contain photoreactive groups. The RNAligand is transformed into a pnhotoreactive ligand by substitution ofphotoreactive nucleic acid nucleotides at specific sites in the ligand.The photochemically active permutations of the initial ligand may bedeveloped through a number of approaches, such as specific substitutionor partial random incorporation of the photoreactive nucleotides.Specific substitution involves the synthesis of a variety ofoligonucleotides with the position of the photoreactive nucleotidechanged manually. The location of the substitution is directed basedupon the available data on binding of the ligand to the target molecule.For example, substitutions are made to the initial ligand in areas ofthe molecule that are known to interact with the target molecule. Forsubsequent selection rounds, the photoSELEX method is used to select forthe ability to crosslink to the target molecule upon irradiation.

In another embodiment of the present invention, nucleic acid ligands areselected by photoSELEX followed by SELEX. PhotoSELEX is performedinitially with oligonucleotide sequences containing photoreactivegroups. Sequences selected for their ability to crosslink to the targetmolecule are then selected for ability to bind the target moleculethrough the SELEX method (Example 13).

In another embodiment of the present invention, a limited selection ofoligonucleotides through SELEX is followed by selection throughphotoSELEX (Example 14). The initial SELEX selection rounds areconducted with oligonucleotides containing photoreactive groups. After anumber of SELEX rounds, photoSELEX is conducted to selectoligonucleotides capable of binding the target molecule.

In yet another embodiment of the present invention, nucleic acid ligandsidentified through SELEX are subjected to limited randomization,followed by selection through photoSELEX (Example 15). SELEX is firstcarried out to completion with nucleic acid sequences not containingphotoreactive groups. The sequence of the nucleic acid ligand is used togenerate a family of oligonucleotides through limited randomization.PhotoSELEX is subsequently performed to select a nucleic acid ligandcapable of photocrosslinking to the target molecule.

In another embodiment of the present invention, photoSELEX is used toidentify a nucleic acid ligand capable of modifying the biologicalactivity of a target molecule (Example 16).

In a further embodiment of the present invention, the photoSELEXmethodology is applied diagnostically to identify unique proteinsassociated with specific disease states (Example 17). In yet anotherembodiment of the present invention, nucleic acid ligands capable ofcrosslinking a target molecule associated with a specific diseasecondition are used in vivo to crosslink to the target molecule as amethod of treating the disease condition (Examples 18 and 19).

In one embodiment of the present invention, RNA ligands identified byphotoSELEX are used to detect the presence of the target protein bybinding to the protein and then photocrosslinking to the protein.Detection may be achieved by incorporating ³²P or ³³P-labels anddetecting material which is retained by a nitrocellulose filter byscintillation counting or detecting material which migrates correctly onan electrophoretic gel with photographic film. Alternatively, photoSELEXcreates a fluorescent chromophore which is detected by fluorescenceemission spectroscopy. Fluorescence emission for the products ofreaction of 5-bromouracil to model peptides (as shown in FIG. 3) hasbeen reported by Dietz and Koch (1987) supra. In another embodiment ofthe invention, a fluorescent label is covalently bound to the RNA anddetected by fluorescence emission spectroscopy. In another embodiment ofthe invention, RNA ligands selected through photoSELEX are used toinhibit the target protein through the same process. In yet anotherembodiment, the photoselected ligand is bound to a support and used tocovalently trap a target.

In a one embodiment of the invention, 5-iodouracil is incorporated intothe RNA sequences of the candidate mixture, and light in the range of320-325 nm is used for irradiation. This combination assuresregionselective photoc-osslinking of the 5-halouracil chromophore to thetarget protein without other non-specific photoreactions. In particular,tryptophan residues of proteins and thymine and uracil bases of nucleicacids are known to be photoreactive. As shown in FIG. 4, 5-iodouracilabsorbs at 325 nm but tryptophan and the standard nucleic acid bases donot. The molar extinction coefficient for 5-iodouracil at 325 nm is 163L/mol·cm. Monochromatic light in the region of 320-325 nm is preferablysupplied by a frequency doubled tunable dye laser emitting at 320 nm orby a helium cadmium laser emitting at 325 nm.

In one embodiment of the invention a xenon chloride (XeCl) excimer laseremitting at 308 nm is employed for the photocrosslinking of5-iodouracil-bearing RNA sequences to a target protein. With this laser,a high yield of photocrosslinking of nucleoprotein complexes is achievedwithin a few minutes of irradiation time.

In another embodiment of the invention, photocrosslinking of5-iodouracil-bearing RNA sequences to a target protein is achieved withwavelength filtered 313 nm high pressure mercury lamp emission or withlow pressure mercury lamp emission at 254 nm absorbed by a phosphor andre-emitted in the region of 300-325 nm. The latter emission is alsocarefully wavelength filtered to remove 254 nm light not absorbed by thephosphor and light in the region of 290-305 nm which could damage theprotein.

In a further embodiment of the invention, photocrosslinking of BrU- orIU-bearing RNA sequences to a target protein is achieved with light inthe region of 350-400 nm which populates directly the triplet state fromthe ground state. Monochromatic light from the third harmonic of aNeodymium YAG laser at 355 nm or the first harmonic from a xenonfluoride (XeF) excimer laser at 351 nm may be particularly useful inthis regard.

In yet another embodiment of the invention the photoreactive nucleotidesare incorporated into single stranded DNAs and amplified directly withor without the photoreactive nucleotide triphosphate.

A. Covalent SELEX and Nucleic Acid Ligands That Bind to HIV-1 RevProtein With and Without Irradiation.

The target protein chosen to illustrate photo-SELEX is Rev from HIV-1.Rev's activity in vivo is derived from its association with theRev-responsive element (RRE), a highly structured region in the HIV-1viral RNA. Previous RNA SELEX experiments of Rev have allowed theisolation of very high affinity RNA ligands. The highest affinityligand, known as “6a,” (SEQ ID NO:5) has a K_(d) of approximately 1 nMand is shown in FIG. 16. The secondary structure of 6a, and itsinteraction with Rev, have been well characterized.

The construction of the nucleic acid library for photo-SELEX was basedupon the Rev 6a sequence (SEQ ID NO:5). During the synthesis of thedeoxyoligonucleotide templates for SELEX, the random region of thetemplate was substituted by a “biased randomization” region, in whichthe ratio of the four input bases was biased in favor of thecorresponding base in the 6a sequence. (Actual ratios were62.5:12.5:12.5:12.5.) For example, if the 6a base for a particularposition is G, then the base input mixture for this synthesis step is62.5% G, and 12.5% of the other three bases.

The photoreactive uracil analogue 5-iodouracil (iU), which has been usedto generate high-yield, region-specific crosslinks betweensingly-substituted iU nucleic acids and protein targets (Willis et al.(1993) Science 262:1255) was used for this example. The iU chromophoreis reactive under long-wavelength ultraviolet radiation, and mayphotocouple to the aromatic amino acids of protein targets by the samemechanism as 5-bromouracil (Dietz et al. (1987) J. Am. Chem. Soc.109:1793). As discussed above, the target for this study is the HIV-1Rev protein, which is necessary for productive infection of the virus(Feinberg et al. (1986) Cell 46:807) and the expression of the viralstructural genes gag, pol and env (Emerman et al. (1989) Cell 57:1155).The interaction of Rev with high affinity RNA ligands is wellcharacterized. A single, high-affinity site within the RRE (the IIBstem) has been identified (Heaphy et al. (1991) Proc. Natl. Acad. Sci.USA 88:7366). In vitro genetic selection experiments have generated RNAligands that bind with high affinity to Rev and have helped determinethe RNA structural elements necessary for Rev:RNA interactions (Bartelet al. (1991) Cell 67:529; Tuerk et al., In the Polymerase ChainReaction (1993); Jensen et al. (1994) J. Mol. Biol. 235:237).

A “biased randomization” DNA oligonucleotide library, based upon thehigh affinity Rev ligand sequence 6a (SEQ ID NO:5), containsapproximately 10¹⁴ unique sequences. This template was used for in vitroT7 transcription with 5-iUTP to generate fully-substituted iU RNA forselection. The photo-SELEX procedure alternated between affinityselection for Rev using nitrocellulose partitioning and monochromatic UVirradiation of the nucleoprotein complexes with denaturingpolyacrylamide gel partitioning of the crosslinked complexes away fromnon-crosslinked RNA sequences. The final procedure utilized asimultaneous selection for affinity and crosslinking using competitortRNA. Each round constitutes a selection followed by the conversion ofrecovered RNA to cDNA, polymerase chain reaction (PCR) amplification ofthe DNA, and in vitro transcription to generate a new pool of iU-RNA. Toamplify RNA's recovered as covalent nucleoprotein complexes, theappropriate gel slice was isolated and proteinase K treated.

The RNA pool was first subjected to three rounds of affinity selectionwith Rev protein, with partitioning of the higher affinity sequences bynitrocellulose filters. Next, the evolving RNA pool was subjected to UVlaser irradiation in the presence of excess Rev protein to allow thoseRNA sequences with the ability to crosslink with the protein to do so.Crosslinked RNA sequences were then partitioned using polyacrylamide gelelectrophoresis (PAGE). These crosslinked RNAs were recovered from thegel material, the linked Rev protein digested away, and the RNAs usedfor cDNA synthesis and further amplification for the next round ofphoto-SELEX. A 308 nm XeCl excimer laser was used for the first round ofphotocrosslinking; thereafter, a 325 nm HeCd laser was employed.

Following four rounds of selection for laser-induced crosslinking, theRNA pool was again put through three rounds of affinity selection.Finally, the RNA pool was selected simultaneously for its ability tobind Rev with high affinity and to crosslink to the protein. This wasaccomplished by using high concentrations of a non-specific nucleic acidcompetitor in the photocrosslinking reaction.

Crosslinked product increased approximately 30-fold from the startingpool to round 13 (FIG. 15). Under these conditions, the greatestincrease in crosslinking is correlated with the greatest increase inaffinity—from round 7 to round 10.

After 13 rounds of selection, the PCR products were cloned and 52isolates sequenced (FIG. 16, SEQ ID NOS:5-55). Class 1 molecules, whichcomprise 77% of the total sequences, contain a very highly conservedmotif, 5′KDAACAN . . . N′UGUUH′M′3′ (SEQ ID NO:56) (FIG. 17). Computerfolding algorithms predict that this conserved motif is base-paired andlies in a stem-loop structure. Subclasses a-d (FIG. 16, SEQ ID NOS:5-43)illustrate different strategies utilized in the “biased randomization”pool to obtain the consensus motif. Class 2 molecules show a highlyconserved 10-base sequence (FIG. 17, SEQ ID NO. 57), which is predictedto fold with the 5′ fixed region of the RNA and forms a structuredistinct from either the class 1 or the 6a (SEQ ID NO:5) motif. Allclass 1 sequences exhibit biphasic binding to Rev, with high affinitydissociation constants (K_(d)s) ranging from 1-10M. Class 2 sequencesshow monophasic binding to Rev with K_(d)s approximately of 30-50 nM.Analysis of round 13 sequences reveal that the frequency of theconsensus motifs for class 1 and class 2 populations was very small inthe starting pool, and some individual sequences arose only through themutational pressures of the photo-SELEX procedure.

Cross-linking behavior differs between the two classes. Under high Revconcentrations (500 nM), and 4 min. of 325 nm irradiation, class 2molecules produce greater crosslink yield and efficiency than class 1molecules (data not shown); presumably, this behavior allows the class 2molecules, with relatively low affinity for Rev, to compete under thephoto-SELEX procedures. For class 1 molecules, longer irradiation timeswill produce higher molecular weight crosslinked species. Although notbound by theory, it is proposed that the RNAs, which contain both anevolved binding domain for Rev, and the fixed regions needed foramplification in SELEX, are able to bind more than one Rev molecule perRNA. Since each RNA contains on average 21 iU bases (RNA length—86bases), it is thought that there is a certain promiscuity of thephotoreaction that allows crosslinking of a single RNA to more than oneRev molecule at high protein concentrations. Class 2 molecules producefewer high molecular weight species upon photocrosslinking; they are, onaverage, iU poor and may contain structures which do not allowbinding/crosslinking to additional Rev molecules.

Analysis of individual round 13 RNAs revealed that a subpopulation couldcrosslink to Rev without laser irradiation. Thus, the single set ofexperiments demonstrated that both covalent SELEX without irradiationand photoSELEX with irradiation can be found in the same system. 4 of 15round 13 sequences analyzed crosslink without laser irradiation (FIG.16). From these few sequences, it was not readily possible to identify asequence motif that confers laser independent crosslinking, although allmolecules considered to date belong to the 1a subclass.

To further investigate laser-dependent and laser-independentcrosslinking (LD-XL and LI-XL, respectively) and avoid the secondaryphotoproducts formed with full-length class 1 molecules, several smallRNAs containing only the evolved sequences were constructed. Trunc2 andtrunc24 (FIGS. 18 and 19) (SEQ ID NOS:58 AND 59) are based upon clones#3 and #24, respectively, and show monophasic binding to Rev with Kds of0.5 nM (trunc2) and 20 nM (trunc24). Trunc2 (FIG. 18) exhibits LD-XLbehavior, and trunc24 (FIG. 19) is capable of both LI-XL and LD-XL.

To explore the conformation and chemical requirements for LD-XL andLI-XL, crosslinking reactions were performed with trunc2 (SEQ ID NO:58)and trunc24 (SEQ ID NO:59) and several Arginine Rich Motif (ARM)proteins. The class of RNA-binding proteins includes the target protein,HIV-1 Rev, and also HIV-1 Tat and the highly similar HIV-2 Rev. LD-XLreactions with trunc2 (FIG. 18, SEQ ID NO:58) show that trunc2 iscapable of crosslinking specifically to both HIV-1 and HIV-2 Revproteins, but not HIV-1 Tat. The two slightly different migratingnucleoprotein complexes probably represent the ability of trunc2 to useone of two iU nucleotides to crosslink the Rev proteins. Although notbound by theory, it is proposed that a tryptophan residue present in thehighly similar ARMs of both Rev proteins is the amino acid necessary forthe specific photo-crosslinking of our high-affinity RNA ligands.

Trunc24 LI-XL (FIG. 19, SEQ ID NO:59), performed with the same proteins,shows crosslinking only to HIV-1 Rev. Like trunc3, trunc24 canphoto-crosslink to HIV-2 Rev (data not shown). It was also observed thatthis LI-crosslink is reversible under highly denaturing conditions, orwith high concentration of nucleic acid competitors. Although not boundby theory, these observations lead to the postulation that LI-XLproceeds by a Michael adduct between the 6 position of an IU and acysteine residue, or possibly a 5 position substitution reaction. Thispostulation is consistent both with the observation that iU undergoesMichael adduct formation. more readily than U, and the fact that HIV-1Rev contains three cysteines, while HIV-2 Rev contains none.

To test for the ability of trunc24 (SEQ ID NO:59) to discriminate HIV-1Rev in a complex mixture, trunc24 and 10 μg of human fibroblast nuclearextract were mixed together with decreasing amounts of HIV-1 Rev (FIG.20). At 50 nM Rev and a 1:100 weight ratio of Rev to nuclear extract, itwas possible to see a very significant crosslinked product betweentrunc24 and Rev. Nuclear extracts and trunc24 alone resulted in nocrosslinked products.

Example 1 describes the synthesis of hairpin RNA oligonucleotides RNA-1(SEQ ID NO:l), RNA-2 (SEQ ID NO:2), and RNA-3 (SEQ ID NO:3) using5-bromouridine triphosphate, 5-iodouridine triphosphate and uridinetriphosphate, respectively. Experiments determining the RNA-proteinbinding curves for RNA-1 (SEQ ID NO:1), RNA-2 (SEQ ID NO:2), and RNA-3(SEQ ID NO:3) to the bacteriophage R17 coat protein are described inExample 2. Example 3 describes the photocrosslinking of the RNAoligonucleotides to the R17 coat protein. The amino acid residue of theR17 coat protein photocrosslinked by RNA-1 (SEQ ID NO:1) afterillumination via xenon chloride (XeCl) excimer laser at 308 nm isdescribed in Example 4. Example 5 describes the photocrosslinking ofiodouracil-substituted RNA-2 (SEQ ID NO:2) to the R17 coat protein bymonochromatic emission at 325 nm. Example 6 describes thephotocrosslinking of RNA-1 (SEQ ID NO:1) and RNA-2 (SEQ ID NO:2) to theR17 coat protein achieved after broad-band emission illumination with atransilluminator. Example 7 describes the photoreaction of 5-iodouracilwith N-acetyltyrosine N-ethyl amide, which appeared to yield aphotocrosslink similar to that achieved with 5-bromouracil-substitutednucleic acids to associated proteins. The preparation of a cDNA from aRNA photocrosslinked to the R17 coat protein is described in Example 8.Example 9 describes the photocrosslinking of an IC-substituted RNAligand to the R17 coat protein.

Example 10 describes the incorporation of halogenated nucleotides intoDNA. Examples 11-15 describes photoSELEX protocols which may be used toproduce specific photoreactive nucleic acid ligands. Example 11describes a continuous photoSELEX method. Example 12 describes a methodin which nucleic acid ligands initially selected through SELEX aresubsequently selected through photoSELEX for the capacity to crosslinkto the target molecule. Example 13 describes one embodiment in whichnucleic acid ligands identified through photoSELEX are then subjected toselection through SELEX and selected for ability to bind the targetmolecule. Example 14 describes another embodiment wherein a limitedSELEX selection is followed by selection through photoSELEX. Example 15describes an embodiment of the present invention in which nucleic acidligands identified through SELEX are subjected to limited randomization,followed by selection through photoSELEX. Example 16 describes a methodfor selecting a nucleic acid ligand capable of modifying the biologicalactivity of a target molecule. Example 17 describes a diagnosticprocedure which uses the SELEX and photoSELEX methods to identifyproteins associated with specific disease processes.

Example 18 describes a method for the in vivo treatment of diseasethrough photoSELEX. A photoSELEX selected nucleic acid ligand able tobind and crosslink to a target molecule associated with a disease stateis introduced into a patient in a number of ways known to the art. Forexample, the photoSELEX ligand may be transiently or constitutivelyexpressed in the appropriate cells of a patient with the disease.Alternatively, the photoSELEX ligand may be taken into a patient's cellsas a double-stranded DNA which is transcribed in the cell in thepresence of iodinated cytosine. Iodinated cytosine may be administeredto the patient, followed by irradiation with X-rays. IC incorporatedinto the nucleic acid ligand synthesized in the appropriate cells allowsthe ligand to crosslink and inactivate the target molecule. Furthermethods of introducing the photoSELEX ligand into a patient includeliposome delivery of the halogenated ligand into the patient's cells.

Example 20 describes the production of modified nucleic acid ligandsthat crosslink, with or without irradiation, to HIV-1 Rev protein. FIG.15 shows the results of crosslinking to the bulk candidate mixture atvarious rounds of SELEX. rd1-round 1 pool RNA; rd7-round 7 pool RNA;rd10-round 10 pool RNA; rd13-round 13 pool RNA; rd13/PK,photocrosslinked round 13 pool RNA proteinase K treated (35 μl of a 100μl reaction was incubated in 0.5% SDS, 50 μg/ml Proteinase K and 1 mMEDTA at 65° C. for one hour); rd13/no iU-round 13 pool RNA transcribedwith UTP (no iU). R-free RNA; XL-crosslinked nucleoprotein complex.

FIG. 16 shows the sequences sequenced after 13 rounds of SELEX (SEQ IDNOS:5-55). The sequences are aligned for maximum homology to the 6asequence (SEQ ID NO:5). Underlines represent potential base pairing asindicated by computer RNA folding algorithms. Dashed underlinesrepresent the 6a ligand “bubble” motif. Sequences flanked by underlinerepresent either loop or bulge regions. Dashes are placed to maximizealignment with 6a. * denotes that two isolates were obtained. +indicateslaser independent crosslinking and −denotes the lack of laserindependent crosslinking to HIV-1 Rev. FIG. 17 (SEQ ID NOS:56-57) showsthe consensus for class 1 and class 2 ligands. FIGS. 18 and 19 show thesequence of Trunc2 (SEQ ID NO:58) and Trunc24 (SEQ ID NO:59) and thespecificity results. 500 nM protein, 20 μg tRNA, and approximately 1 nMof kinased trunc2 RNA were incubated for 10 min. at 37° C. andirradiated for 4 min. at 325 nm. t2-trunc2 RNA irradiated without addedprotein; t2Rev1/O′-trunc2 RNA, HIV-1 Rev protein and 0 min. ofirradiation; t2/Rev1/4′-trunc2 RNA, HIV-1 Rev protein, and 4 min. ofirradiation; t2/Rev1/4′/PK-trunc2 RNA, HIV-1 Rev protein, 4 min. ofirradiation, and proteinase K treated as in FIG. 1; t2/Rev2/4′-trunc2RNA, HIV-2 Rev protein, and 4 min. of irradiation; t2/Tat/4′-trunc2 RNA,HIV-1 Tat protein, and 4 min. of irradiation. R-free RNA; XL-crosslinkednucleoprotein complex. FIG. 20 shows the trunc24 photoindependentcrosslinking with HIV-1 Rev in the presence of human nuclear extract.

II. Solution SELEX.

This embodiment of the present invention presents several improvedmethods for partitioning between oligonucleotides having high and lowaffinity for a target molecule. The method of the present invention hasseveral advantages over prior art methods of partitioning: (1) it allowsthe isolation of nucleic acid ligands to the target without alsoisolating nucleic acid ligands to the partitioning matrix; (2) itincreases the speed and accuracy by which the oligonucleotide candidatemixture is screened; and (3) the solution SELEX procedure can beaccomplished in a single test tube, thereby allowing the partitioningstep to be automated.

The materials and techniques required by the method of the presentinvention are commonly used in molecular biology laboratories. Theyinclude the polymerase chain reaction (PCR), RNA or DNA transcription,second strand DNA synthesis, and nuclease digestion. In practice, thetechniques are related to one another in a cyclic manner as illustratedin FIG. 21.

In the SELEX method, described by Tuerk and Gold (1990) Science 249:1155and illustrated in FIG. 21, a single-stranded nucleic acid candidatemixture is generated by established procedures on a nucleic acidsynthesizer, and is incubated with dNTP and Klenow fragment to generatea population of double-stranded DNA templates. The double-stranded DNAor the RNA transcribed from them are purified, and contacted with atarget molecule. RNA sequences with enhanced affinity to the targetmolecule form nucleic acid-target complexes. This is followed bypartitioning of bound and unbound nucleic acids, and separation of thetarget molecule from the bound nucleic acids. cDNA is synthesized fromthe enhanced affinity nucleic acids and double-stranded DNA generated byPCR amplification. The cycle is repeated until the complexity of thecandidate mixture has decreased and its affinity as well as specificityto the target has been maximized.

A novel feature of the solution SELEX method is the means by which thebound and free members of the nucleic acid candidate mixture arepartitioned. In one embodiment of the method of the present invention,generation of two physically distinct cDNA pools is accomplished by useof primer extension inhibition. One cDNA extension step is added to thebasic SELEX protocol between steps 2 and 3 above, which allows thegeneration of two physically distinct cDNA pools—one having highaffinity for the target and one having low affinity for the target—whichare easily distinguished and separated from each other. Primer extensioninhibition analysis is a common technique for examining site-boundproteins complexed to nucleic acids (Hartz et al. (1988) MethodsEnzymol. 164:419), and relies on the ability of high affinity complexesto inhibit cDNA synthesis. Examples of protein-nucleic acid interactionsstudied by primer extension inhibition include ribosome binding to themRNA ribosome-binding site (Hartz et al. (1988) Meth. Enzym. 164:419) aswell as binding of the unique E. coli translation factor, SELB protein,to the mRNA selenocysteine insertion sequence (Baron et al. (1993) Proc.Natl. Acad. Sci. USA 90:4181).

In one embodiment of the solution SELEX scheme, the first cDNA extensionis performed in the presence of chain terminating nucleotidetriphosphates. Under these conditions, oligonucleotides with lowaffinity for the target which form fast dissociating complexes with thetarget are converted into truncated cDNAs with a 3′-end terminated witha nonextendible nucleotide. The truncated cDNA chain is unable to annealto the PCR primers, and therefore, is non-amplifiable. In contrast,tight complexes formed between high affinity oligonucleotides and thetarget molecule, characterized by slow dissociation rates, inhibit cDNAextension. The chain terminators are not incorporated into the nascentcDNA chain synthesized from the high affinity oligonucleotide becausecDNA synthesis is blocked by the tightly bound target molecule. Fulllength CDNA from the high affinity complexes are obtained during asecond round of cDNA extension in which the target and chain terminatorshave been removed from the system. Thus, weak affinity complexes areconverted into truncated cDNA lacking the primer annealing site whiletight complexes are converted into full length cDNA and are amplified byPCR (FIG. 22). The stringency of this method is easily modified byvarying the molar ratio of chain terminators and dNTPs or theconcentration of the polymerase, as primer extension inhibition issensitive to polymerase concentration (Ringquist et al. (1993)Biochemistry 32:10254). As used in the present disclosure, the term“stringency” refers to the amount of free RNA that will be convertedinto PCR product.

Therefore, one crucial feature of the invention is its ability topartition strong and weak affinity complexes into amplifiable andnon-amplifiable nucleic acid pools without requiring a partitioningmatrix. It is the unique properties of these cDNA pools that allowselective amplification of the high affinity ligand.

The target molecule can be a protein (either nucleic acid or non-nucleicacid binding protein), nucleic acid, a small molecule or a metal ion.The solution SELEX method allows resolution of enantiomers as well asthe isolation of new catalytic nucleic acids.

Primer extension inhibition may be achieved with the use of any of anumber of nucleic acid polymerases, including DNA or RNA polymerases,reverse transcriptase, and Qβ-replicase.

The candidate mixture of nucleic acids includes any nucleic acid ornucleic acid derivative, from which a complementary strand can besynthesized.

Prior art partitioning included use of nitrocellulose or an affinitycolumn. One disadvantage of the prior art partitioning was thephenomenon of matrix binders in which nucleic acids that specificallybind the partitioning matrix are selected along with those thatspecifically bind the target. Thus, one advantage of the method of thepresent invention is that it overcomes unwanted selective pressureoriginating with use of a partitioning matrix by only using suchmatrixes after nucleic acids with high affinity for the target have beenpartitioned in solution and amplified. Moreover, the ability topartition strong and weak affinity complexes during cDNA synthesis,based on the ability of only the strongest complexes to inhibitextension by a polymerase, results in the selection of only the highestaffinity nucleic acid ligands. It is estimated that complexes withdissociation constants in the nanomolar or less range will efficientlyblock cDNA synthesis. The method of the present invention is expected topreferentially screen nucleic acid candidate mixtures for members thatbind the target at this limit.

The use of primer extension inhibition allows partitioning of theoligonucleotide candidate mixture into two pools—those oligonucleotideswith high target affinity (amplifiable) and those with low targetaffinity (non-amplifiable). As described above, chain terminators may beused to poison the first extension product, rendering the low affinitycDNAs non-amplifiable.

In another embodiment of the method of the present invention,restriction enzymes are used to selectively digest the cDNA generatedfrom the low affinity nucleic acids. A number of restriction enzymeshave been identified that cleave single-stranded DNA. These enzymescleave at specific sequences but with varying efficiencies. Partitioningof weak and strong affinity nucleic acids is accomplished by primerextension in the presence of the four dNTPs, followed by removal of thetarget and a second extension with modified nucleotides that areresistant to enzymatic cleavage. The cDNA pools can then be incubatedwith the appropriate restriction enzyme and the cDNA synthesized duringthe first extension cleaved to remove the primer annealing site andyield a non-amplifiable pool. Increased efficiency of cleavage isobtained using a hairpin at the restriction site (RS) to create alocalized double-stranded region (FIG. 24).

In another embodiment of method of the present invention, cDNA sequencescorresponding to low affinity nucleic acids are rendered selectivelydegradable by incorporation of modified nucleotide into the first cDNAextension product such that the resulting cDNA is preferentiallydegraded enzymatically or chemically.

In another embodiment of the method of the present invention, the firstextension product can be removed from the system by an affinity matrix.Alternatively, the matrix could be used to bind the second extensionproduct, e.g., the cDNAs corresponding to high affinity nucleic acids.This strategy relies on the incorporation of modified nucleotides duringcDNA synthesis. For instance, the first cDNA extension could beperformed in the presence of modified nucleotides (e.g., biotinylated,iodinated, thiolabelled, or any other modified nucleotide) that allowretention on an affinity matrix (FIG. 25). In an alternate embodiment ofthe method of the present invention, a special sequence can also beincorporated for annealing to an affinity matrix. Thus, first synthesiscDNAs can be retarded on commercially obtainable matrices and separatedfrom second synthesis cDNA, synthesized in the absence of the modifiednucleotides and target.

In another embodiment of the invention, exonuclease hydrolysisinhibition is used to generate a pool of high affinity double-strandednucleic acid ligands.

In yet another embodiment of the invention, the solution SELEX method isused to isolate catalytic nucleic acids.

In another embodiment of the invention, solution SELEX is used topreferentially amplify single-stranded nucleic acids.

In a further embodiment of the invention, the solution SELEX method isautomated.

Removal of the target to allow cDNA synthesis from the high affinitynucleic acids can also be accomplished in a variety of ways. Forinstance, the target can be removed by organic extraction or denaturedby temperature, as well as by changes in the electrolyte content of thesolvent. In addition, the molecular repertoire of the candidate mixturethat can be used with the invention include any from which a secondcomplementary strand can be synthesized. Single-stranded DNA as well asRNA can be used, as can a variety of other modified nucleotides andtheir derivatives.

The following non-limiting examples illustrate the method of the presentinvention. Example 21 describes the solution SELEX process whereinpartitioning between high and low affinity nucleic acids is achieved byprimer extension inhibition. Example 22 illustrates the solution SELEXprocess wherein partitioning is achieved by restriction enzyme digestionof low affinity RNA. Example 23 describes the solution SELEX processwherein low affinity nucleic acids are separated from high affinitynucleic acids by affinity chromatography. Example 24 describes theisolation of high affinity double-stranded nucleic acid ligands with theuse of exonuclease inhibition. Example 25 describes the isolation ofcatalytic nucleic acids. Example 26 describes an automated solutionSELEX method.

The examples provided are non-limiting illustrations of methods ofutilizing the present invention. Other methods of using the inventionwill become apparent to those skilled in the art from the teachings ofthe present disclosure.

EXAMPLE 1

Synthesis of RNA Sequences RNA-1, RNA-2, RNA-3, and RNA-7 and R17 CoatProtein.

RNA-1 (SEQ ID NO:1), RNA-2 (SEQ ID NO:2), and RNA-3 (SEQ ID NO:3) shownin FIG. 6 and RNA-7 (SEQ ID NO:4) shown in FIG. 12 were prepared by invitro transcription from synthetic DNA templates or plasmids usingmethodology described by Milligan and co-workers (Milligan et al. (1987)Nucleic Acids Res. 15:8783). Transcription reactions contained 40 mMtris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 8.1 at 37°C.), 1 mM spermidine, 5 mM dithiothreitol (DTT), 50 μg/ml of bovineserum albumin (BSA), 0.1% (v/v) Triton X-100, 80 mg/ml of polyethyleneglycol (m_(r) 8000), and 0.1 mg/ml of T7 RNA polymerase. Largerquantities of RNA were prepared with 3-5 mM of each of the nucleotidetriphosphates (NTPs), 25 mM magnesium chloride, and 1 μM DNA template or0.1 μg/ml of plasmid. Body-labeled RNAs were prepared in 100 μMreactions with 1 mM each of the three NTPs, 0.25 mM of the equivalentradiolabelled NTP ([α-³²P] NTP, 5 μCi), 15 mM MgCl₂, and 0.1 mg/ml of T7RNA polymerase. Nucleotides, including 5-iodouridine triphosphate and5-bromouridine triphosphate, were obtained from Sigma Chemical Co., St.Louis, Mo. RNA fragments were purified by 20% denaturing polyacrylamidegel electrophoresis (PAGE). The desired fragment was eluted from thepolyacrylamide and ethanol-precipitated in the presence of 0.3 M sodiumacetate. R17 bacteriophage was propagated in Escherichia coli strainS26, and the coat protein was purified using the procedure described byCarey and coworkers (Carey et al. (1983) Biochemistry 22:4723).

EXAMPLE 2

Binding Constants for RNA-1 and RNA-2 to R17 Coat Protein.

RNA-protein binding curves for hairpin variants RNA-1 (SEQ ID NO:1),RNA-2 (SEQ ID NO:2) and RNA-3 (SEQ ID NO:3) to the bacteriophage R17coat protein are shown in FIG. 7. The association constants between coatprotein and the RNA hairpin variants were determined with anitrocellulose filter retention assay described by Carey and co-workers(Carey et al. (1983) supra). A constant, low-concentration of³²P-labeled RNA was mixed with a series of coat protein concentrationsbetween 0.06 nM and 1 μM in 10 mM magnesium acetate, 80 mM KCI, 80 μg/mlBSA, and 100 mM Tris-HCl (pH 8.5 at 4° C.) (TMK buffer). These were thesame solution conditions used in the crosslinking experiments. Afterincubation at 4° C. for 45-60 min, the mixture was filtered through anitrocellulose filter and the amount of complex retained on the filterdetermined by liquid scintillation counting. For each experiment thedata points were fit to a non-cooperative binding curve and the K_(d)value shown in FIG. 7 was calculated.

EXAMPLE 3

Photocrosslinking of RNA-1 and RNA-2 to R17 Coat Protein at 308 nm.

³²P-Labeled RNA sequences RNA-1 (SEQ ID NO:1) and RNA-2 (SEQ ID NO:2) (5nM) and R17 coat protein (120 nM) were each incubated on ice in 100 mMTris-HCl (pH 8.5 at 4° C.), 80 mM KCl, 10 mM magnesium acetate, 80 μg/mlof BSA for 15-25 min before irradiations. These are conditions underwhich the RNA is fully bound to the coat protein. The RNAs were heatedin water to 85C for 3 min and quick cooled on ice before use to ensurethat the RNAs were in a hairpin conformation (Groebe and Uhlenbeck(1988) Nucleic Acids Res. 16:11725). A Lambda Physik EMG-101 excimerlaser charged with 60 mbar of xenon, 80 mbar of 5% HCl in helium and2360 mbar of helium was used for 308 nm irradiations. The output of theXeCl laser was directed unfocused toward a 4 mm wide by 1 cm path lengthquartz cuvette containing the RNA-protein complex. The laser wasoperated in the range of 60 mJ/pulse at 10 Hz; however, only about 25%of the laser beam was incident upon the reaction cell. Photocrosslinkingyields of RNA-1 (SEQ ID NO:1) and RNA-2 (SEQ ID NO:2) to the R17bacteriophage coat protein as a function of irradiation time are shownin FIG. 8. Crosslinked RNA was separated from uncrosslinked RNA by PAGE,and the yields were determined by autoradiography. Crosslinking of5-bromouracil-containing variant RNA-1 (SEQ ID NO:1) maximized at about40% because of competitive photodamage to the coat protein whichinhibits binding to the RNA (Gott et al. (1991) supra). Less photodamageto coat protein occurred with RNA 2 because of the shorter irradiationtime.

Crosslinking as a function of photons absorbed indicated that thequantum yield for crosslinking of BrU-RNA 1 is 0.014 and forcrosslinking of IU-RNA-2 (SEQ ID NO:2), 0.006 with irradiation at 308nm. In spite of the lower quantum yield, a higher crosslinking yield wasobtained with IU-RNA 2 because of the seven times higher absorptionprobability of the IU chromophore at 308 nm. BrU and IU absorb at 308 nmwith molar extinction coefficients of 385 and 2640 L/mol·cm,respectively. Hence, a high level of photocrosslinking of the IU-RNA wasachieved prior to protein damage.

EXAMPLE 4

Identification of the Amino Acid Residue Involved in the Crosslink ofRNA-1 to R17 Coat Protein.

Large Scale Crosslinking of RNA-1 (SEO ID NO:1) to R17 Coat Protein.

A 10 ml solution containing 300 nM 5′-end-labeled RNA and 500 nM coatprotein was incubated on ice in the presence of 100 mM Tris-HCl (pH 8.5at 4° C.), 10 mM Mg(OAc)₂, 80 mM KCI, 80 mg bovine serum albumin (BSA),and 5 mM dithiothreitol (DTT) for 10-90 min. A Lambda Physik EMG-101excimer laser was used for monochromatic irradiation at 308 nm. The beamoutput was measured at 69±5 mJ/pulse at 10 Hz. Approximately 50% of thebeam was focused through a 7 mm-diameter circular beam mask into a 1 cmpath length quartz cuvette in a thermostated cell holder. The laserpower was measured with a Scientech 360-001 disk calorimeter powermeter. The temperature was regulated at 4±2° C. with a Laude RC3circulating batn.

The 10 ml reaction mixtures were prepared just prior to the irradiationswhich were performed in 2 ml fractions. After 5 min of irradiation theprotein concentration was brought to 1 μM. The reaction mixture was thenincubated for 3 min to allow exchange of photodamaged protein for freshprotein in the nucleoprotein complex and irradiated for an additional 5min. This step was repeated nine times to give 90 ml of irradiatedsample. The crosslinking, analyzed by 20% denaturing PAGE, andquantitated on a Molecular Dynamics Phosphoimager, revealed 22%crosslinking.

The 90 ml sample contained 5.9 nmol of crosslinked RNA, 21 nmol of freeRNA, 97 nmol of free coat protein, and 7.2 mg of BSA. The total volumewas reduced to 20 ml and split equally between two 50 ml polypropylenescrew cap centrifuge tubes (Nalgene) and ethanol precipitated overnightat −20° C. The RNA and proteins were spun down to a pellet at 13,000 rpmin a fixed angle J-20 rotor with an Beckman J2-21 centrifuge. Eachpellet was resuspended in 1 ml of 0.5 M urea, 50 mM Tris-HCl pH 8.3, and0.2% SDS for 48 h at 4° C. with shaking. The fractions were combined,and the SDS was then removed by precipitation so as not to decrease theactivity of trypsin. This was achieved using 40 mM KCl, and theprecipitate was removed by spinning through a 0.22 μm cellulose acetatespin filter. The trypsin conditions were optimized using 500 μl of thesolution.

Proteolytic Digestion.

The remaining 1.5 ml of crosslinked RNA solution containing free RNA andprotein was brought to 6 ml to contain 1 M urea, 20 mM CaCl₂, and 6 mMDTT, and then 1.61 mg (1:5 w/w.) trypsin-TPCK (251 units/mg) was added.The reaction proceeded at 36° C. for. 2 h at which time 1.61 mg moretrypsin was added. At 4 h a 100 μL aliquot was removed and the reactionstopped by quick freezing. The reaction was analyzed by 20%polyacrylamide 19:1 crosslinked, 7 M urea, 90 mM Tris-borate/2 mM EDTA(TBE) gel electrophoresis (20% urea denaturing PAGE).

Purification of the digested crosslinked RNA.

The trypsin reaction mixture was brought to 10 ml to reduce the molarconcentration of salt, and run through a 240 μl DEAE ion exchangecentrifuge column. The column was washed with 100 mM NaCl and spun dryin a bench top centrifuge to remove free peptide. The column boundmaterial containing the RNA and crosslinked tryptic fragment was elutedfrom the column with 1 ml of 600 mM NaCl and the column spun dry. Anadditional 200 μl of 600 mM NaCl was spun through the column. The twofractions were pooled, ethanol precipitated and pelleted at 10,000 rpmfor 35 min at 4° C. The pellet was resuspended in 25 μl of 7 M urea-TBEbuffer, 10 mM DTT, 0.1% bromophenol blue, 0.1% xylene cylanol, andheated to 85° C. for 4 min and purified by 20% denaturing PAGE. The gelran for 3.5 h at 600 V. A 5 min phosphoimage exposure was taken of thegel. The digested protein-RNA crosslink was then electrolyticallyblotted from the gel onto a PVDF protein sequencing membrane (0.2micron) from Bio-RAD. The membrane was air dried, coomassie stained for1 min, destained for 2 min in 50% MeOH: 50% H₂O, and rinsed twice withdeionized H₂O. An autoradiogram was made of the membrane to visualizethe digested protein RNA crosslink which was excised from the membraneand submitted for Edman degradation. The immobilized peptide wassequenced by automated Edman degradation, performed on an AppliedBiosystems 470A sequencer using manufacturer's methods and protocols(Clive Slaughter, Howard Hughes Medical Institute, University of Texas,Southwestern). The Edman analysis indicated that the position of thecrosslink was tyrosine-85 based upon the known amino acid sequence(Weber (1983) Biochemistry 6:3144).

EXAMPLE 5

Photocrosslinking of RNA-2 to R17 Coat Protein at 325 nm.

In an experiment analogous to that described in Example 3,IU-substituted RNA-2 (SEQ ID NO:2) was photocrosslinked to R17 coatprotein with monochromatic emission at 325 nm from an Omnichrome HeCdlaser (model 3074-40M325). The power output of the HeCd laser was 37 mWand the total beam of diameter 3 mm was incident upon the sample. Toincrease excitation per unit time the beam was reflected back throughthe sample with a dielectric-coated concave mirror. Crosslinked RNA wasseparated from uncrosslinked RNA by PAGE, and the yields were determinedwith a Phospholmager. The percent of the RNA crosslinked to the proteinas a function of irradiation time is shown in FIG. 9. High-yieldcrosslinking occurred without photodamage to the R17 coat protein. In aseparate experiment analogous irradiation of coat protein alone at 325nm with yet a higher dose resulted in protein which showed the samebinding constant to R17 coat protein. Irradiation at 325 nm ofBrU-containing RNA-1-R17 coat protein complex did not result incrosslinking because the BrU chromophore is transparent at 325 nm.

EXAMPLE 6

Photocrosslinking of RNA-1 and RNA-2 to R17 Coat Protein with aTransilluminator.

In an experiment analogous to that described in Example 3, RNA-1 (SEQ IDNO:1) and RNA-2 (SEQ ID NO:2) were photocrosslinked to the R17 coatprotein with broad-band emission in the range of 312 nm from a FisherBiotech Transilluminator (model FBTIV-816) filtered with polystyrene.Crosslinked RNA was separated from uncrosslinked RNA by PAGE, and theyields were determined by autoradiography. Percent RNAs crosslinked toprotein as a function of irradiation time is shown in FIG. 10.

EXAMPLE 7

Photoreaction of 5-Iodouracil with N-Acetyltyrosine N-Ethyl Amide.

N-acetyltyrosine N-ethylamide was prepared as described by Dietz andKoch (1987) supra. Irradiation of a pH 7, aqueous solution of iodouraciland 10 mol equivalent excess of N-acetyltyrosine N-ethyl amide at 308 nmwith a XeCl excimer laser gave a photoadduct identical to thephotoadduct (structure 6) from irradiation of bromouracil andN-acetyltyrosine N-ethylamide (Dietz and Koch (1987) supra) as shown inFIG. 11. Product comparison was performed by C-18 reverse phase HPLC andby ¹H NMR spectroscopy. Although little is known about the mechanism ofphotocrosslinking of IU-substituted nucleic acids to associatedproteins, this result suggests that it is similar to that ofphotocrosslinking of BrU-substituted nucleic acids to associatedproteins.

EXAMPLE 8

Preparation of a cDNA from an RNA Photocrosslinked to a Protein.

RNA-7 (SEQ ID NO:4) (FIG. 11) was prepared using methodology as reportedin Example 1 using a plasmid instead of a DNA template. Thephotocrosslinking was performed as described in Example 3. A 4 mlreaction mixture consisting of 6.75 M RNA and 120 nM R17 coat proteinwas irradiated, 2 ml at a time, at 308 nm with unfocused emission from aXeCl excimer laser. The laser produced So mJ/pulse and was operated at10 Hz. The reaction proceeded to near quantitative crosslinking, 85-90%,in 5 min of irradiation. After crosslinking, 1 ml of the total reactionmixture was removed; EDTA (80 mM), SDS (0.1%), and CaCl₂ (0.1 mM) wereadded; the free (unbound) RNA present was purified away; and the proteindigested with Proteinase K at 60° C. for 30 min. The RNA bound toresidual protein was ethanol precipitated to remove salts and spun to apellet. The pellet was washed three times with 70% ethanol to remove anyresidual salts. A reverse transcription reaction was employed to make acomplementary DNA copy of the RNA template. A 13-base promoter wasannealed to the RNA and the reverse transcription reaction was performedunder the standard conditions of the manufacturer, Gibco (Gaithersburg,Md.), and was stopped after 1 hr. The cDNA was body labelled with³²P-labelled deoxycytidine triphosphate. The RNA template was thenremoved by hydrolyzing with 0.2 M sodium hydroxide at 100° C. for 5 min.The formation of the cDNA was followed by PAGE. A hydrolysis ladder andmarkers were added to the gel to determine the length of the cDNA. ThecDNA co-migrated with the 44 nucleotide RNA template. If there had beena stop in the cDNA as a result of crosslinking modification, a shortenedproduct of 31 nucleotides would have been observed. A small amount of astop product was observed in the 22-25 nucleotide region of the gel, butthis may have resulted from the hairpin secondary structure which beginsat position 25 of the cDNA on the RNA template. No stop in the 31nucleotide region of the gel appeared; this established that the reversetranscriptase had read through the position of the crosslink. A diagramof the gel appears in FIG. 14.

EXAMPLE 9

Iodocytosine Photocrosslinking.

5-iodocytosine (IC) was incorporated in a hairpin RNA (RNA 8) thatcontained cytosine at the −5 position and bound the R17 coat proteinwith high affinity. The IC-bearing RNA is designated RNA 9. RNA 9 (5 nM)and R17 coat protein (120 nM) were incubated on ice in 100 mM Tris-HCl(pH 8.5 at 4° C.)/80 mM KCl/10 mM magnesium acetate/80 μg/ml BSA for15-25 min prior to irradiation. The RNA in water was heated to 85° C.for 3 min and quick cooled on ice before use to ensure that it would bein a conformation that bound the coat protein (Groebe and Uhlenbeck(1988) supra). The complex was irradiated for 5 min at 4° C., and theexperiment was compared to control irradiations of RNA 2 and RNA 8 coatprotein complexes.

Irradiation of RNA 8-coat protein complex resulted in no crosslinkedproduct. Irradiation of RNA 9-coat protein complex resulted in theformation of a crosslink that formed in high yield (70-80%) similar tothe yield of the control irradiation of RNA 2-coat protein complex(80-90%) crosslinking of RNA 9 is presumed to occur through a similarmechanism as RNAs containing IU at position −5 of the loop hairpin(FIGS. 6 and 12). This assumption is based on the specificity of thecrosslink since RNA 8 did not photocrosslink.

EXAMPLE 10

Incorporation of Halogenated Nucleotides into DNA Ligands.

Photoreactive nucleotides may be incorporated into a DNA ligand capableof crosslinking to a target molecule upon irradiation by the methodsdiscussed above. 5-Bromodeoxyuracil (BrdU), 8-bromo-2′-deoxyadenine, and5-iodo-2′-deoxyuracil are examples of such photoreactive nucleotides.

EXAMPLE 11

PhotoSELEX.

In one embodiment of the present invention, the photoSELEX method isapplied to completion in the selection of a nucleic acid ligand whichbinds and photocrosslinks to a target molecule.

A randomized set of nucleic acid oligonucleotides is synthesized whichcontain photoreactive groups. The oligonucleotides of the candidatemixture may be partially or fully saturated at each available positionwith a photoreactive group. The candidate mixture is contacted with thetarget molecule and irradiated at the appropriate wavelength of light.Oligonucleotides crosslinked to the target molecule are isolated fromthe remaining oligonucleotides and the target molecule removed. cDNAcopies of the isolated RNA sequences are made and amplified. Theseamplified cDNA sequences are transcribed into RNA sequences in thepresence of photoreactive groups, and the photoSELEX process repeated asnecessary.

EXAMPLE 12

Selection of Enhanced Photocrosslinking Ligands: SELEX Followed byPhotoSELEX.

In one embodiment of the method of the present invention, selection ofnucleic acid ligands through SELEX is followed by selection throughphotoSELEX for ligands able to crosslink the target molecule. Thisprotocol leads to ligands with high binding affinity for the targetmolecule that are also able to photocrosslink to the target.

Photoreactive nucleotides are incorporated into RNA by T7 polymerasetranscription with the reactive nucleotide triphosphate in place of aspecified triphosphate. For example, 5-bromouridine triphosphate issubstituted for uridine triphosphate or 8-bromo-adenosine triphosphateis substituted for adenosine triphosphate. A randomized set of RNAsequences containing photoreactive nucleotides are generated and theSELEX methodology applied. The initial SELEX rounds are used toeliminate intrinsically poor binders and enhance the pool of moleculesthat converge to form a pool of RNAs that contain the photoreactivegroup(s) and which bind to the target molecule. Aliquots from theinitial SELEX rounds are irradiated and the enhancement ofphotocrosslinking followed via PAGE as the rounds proceed. As a slowermigrating band representing crosslinked products starts to becomeevident, the pool of RNAs are introduced into rounds of photoSELEX. RNAsthat have a photoreactive group adjacent to a reactive amino acidresidue in the nucleoprotein complexes form a crosslink and are selectedand RNAs that do not have reactive nucleotides in proximity to reactivetarget residues are eliminated.

This protocol selectively applies photoSELEX selection to previouslyidentified ligands to a target molecule.

EXAMPLE 13

PhotoSELEX Followed by SELEX.

In another embodiment of the method of the present invention, an RNAligand able to photocrosslink a target molecule is preselected throughthe photoSELEX methodology. Subsequently, SELEX is performed to select acrosslinking oligonucleotide for ability to bind the target molecule.

EXAMPLE 14

Limited SELEX Followed by PhotoSELEX.

In this embodiment of the present invention, nucleic acid ligands areselected through the SELEX process for a limited number of selectionrounds. SELEX is not applied to completion as in Example 12. Rather, thecandidate mixture is partially selected for oligonucleotides havingrelatively enhanced affinity for the target molecule. The randomoligonucleotides of the candidate mixture contain photoreactive groupsand the initial SELEX selection is conducted in the absence ofirradiation. PhotoSELEX is then performed to select oligonucleotidesable to crosslink to the target molecule.

This protocol allows the selection of crosslinking ligands from a poolof oligonucleotides with a somewhat enhanced capacity to bind the targetmolecule and may be useful in circumstances where selection tocompletion through SELEX does not yield crosslinking ligands.

EXAMPLE 15

Limited Directed PhotoSELEX.

In one embodiment of the method of the present invention, in whichnucleic acid ligands identified through SELEX are subjected to limitedrandomization, followed by selection through photoSELEX.

The construction of the DNA template used to transcribe the partiallyrandomized RNA is based on the sequence of the initially selected ligandand contains at each position primarily the nucleotide that iscomplementary to that position of the initial selected RNA sequence.However, each position is also partially randomized by using smallamounts of each of the other three nucleotides in the sequencer, whichvaries the original sequence at that position. A limited RNA pool isthen transcribed from this set of DNA molecules with a photoreactivetriphosphate replacing a specific triphosphate in the reaction mix(i.e., BrU for U). The partially randomized set of RNA molecules whichcontains the photoreactive nucleotides is mixed with a quantity of thetarget protein. Bound RNAs that have a photoreactive group adjacent to areactive amino acid residue in the nucleoprotein complex form covalentcrosslinks upon irradiation. RNAs that bind and crosslink are selectedthrough several rounds of photoSELEX and separated away from RNAs thatbind but do not crosslink.

EXAMPLE 16

Methods for Modifying a Target Molecule.

In another embodiment of the method of the present invention, photoSELEXis applied to develop a ligand capable of modifying a target molecule.Under these circumstances, incorporation of a photoreactive group ontoor into a ligand selected by photoSELEX or SELEX may modify the targetin several ways such that the biological activity of the target moleculeis modified. For example, the target molecule may be inactivated byphotocrosslinked ligand. Mechanisms of inactivation include electron orhydrogen abstraction from the target molecule or radical addition to thetarget molecule that elicit a chemical modification. These differentmechanisms may be achieved by changing the mode of irradiation.

A ligand selected through photoSELEX used as a diagnostic for a targetmolecule with ultraviolet (UV) light may also inactivate the same targetin vivo if the source of irradiation is changed to X-rays or gamma rays.The resultant vinyl radical may work similarly to a hydroxyl radical,that is, by abstraction of hydrogen atoms from the binding domain of thetarget molecule.

X-ray irradiation of the R17 coat protein bound to radio-labelled IU- orBrU-substituted RNA hairpin sequences may result in the formation of acrosslink. The BrU or IU chromophore may also be excited to a higherenergy state by X-ray irradiation resulting in the formation of a vinylradical (Mee (1987) in: Radiation Chemistry: Principles and Applications(Farhataziz and Rodgers, eds.), VCH Publishers, New York, pp. 477-499).The radical abstracts a hydrogen from the binding domain of the R17 coatprotein, thereby reducing or inhibiting its ability to bind the RNAligand. Inactivation is tested by X-ray irradiation of the R17 coatprotein in the presence and absence of substituted RNAS. The formationof crosslinked complexes is analyzed by PAGE. The effect of X-rayirradiation of RNA resulting in modification of binding by modificationof the protein domain is followed by nitrocellulose binding assay.

EXAMPLE 17

Diagnostic Use of PhotoSELEX To Identify Unique Proteins Associated withSpecific Disease Processes.

A goal of diagnostic procedures is to correlate the appearance of uniqueproteins with specific disease processes. Some of these correlations areobvious, e.g., after bacterial or viral infections, one can detectantigens which are antigen specific or antibodies to such antigens notfound in the blood of uninfected subjects. Less obvious correlationsinclude the appearance in serum of α-foeto protein which is directlycorrelated with the presence of the most common form of testicularcancer.

The photoSELEX method may be applied to the discovery of heretoforeunknown correlations between biological proteins and important humandiseases. In one embodiment of the present invention, serum is takenfrom a patient with a disease, RNA ligands to all the proteins in theserum are produced and adsorbed to normal sera. RNA ligands to serumproteins may be identified through the SELEX method, with subsequentincorporation of photoreactive groups, or may be identified throughphoto-SELEX, initially selected from a candidate mixture ofoligonucleotides containing one or more photoreactive groups. RNAligands left unbound are those which specifically bind only uniqueproteins in the serum from patients with that disease. For example, RNAligands are initially identified to a limited number of serum proteins(e.g., 11). The RNA ligands identified contain a modified NTP having areversible or photoreactive functional group capable of crosslinkingreversibly or non-reversibly with the target protein. Optionally, thepresence of a cross-linked ligand to every protein may be verified. TheRNA ligands are then removed and amplified. RNA is then transcribed fora second SELEX round. RNA is now bound to a large excess of 10 of theoriginal 11 proteins, leaving an RNA ligand specific for the unique(11th) protein. This RNA is then amplified. This is a subtractivetechnique.

In one embodiment of the diagnostic method of the present invention, themethod described above is used to identify a ligand to an abnormalprotein, for example, an α-foeto protein. Sera from patients withimportant diseases is obtained and RNA ligands to all proteins presentidentified. The RNA ligands are adsorbed to normal sera, leaving anunbound ligand. The unbound ligand is both a potential diagnostic agentand a tool for identifying serum proteins specifically associated with adisease.

EXAMPLE 18

Method of Treating Disease by In Vivo Use of Photocrosslinking NucleicAcid Ligand.

A nucleic acid ligand to a target molecule associated with a diseasestate is selected through the photoSELEX process (Example 11). ThephotoSELEX selected nucleic acid ligand may be introduced into a patientin a number of ways known to the art. For example, the non-halogenatedphotoSELEX ligand is cloned into stem cells which are transferred into apatient. The ligand may be transiently or constitutively expressed inthe patient's cells. IC administered to the patient is incorporated intothe oligonucleotide product of the cloned sequence. Upon irradiation,the ligand is able to crosslink to the target molecule. Irradiation mayinclude visible, 325 nm, 308 nm, X-ray, ultraviolet, and infrared light.

Alternatively, the photoSELEX ligand may be taken into a patient's cellsas a double-stranded DNA which is transcribed in the cell in thepresence of iodinated cytosine. Further methods of introducing thephotoSELEX ligand into a patient include liposome delivery of thehalogenated ligand into the patient's cells.

EXAMPLE 19

PhotoSELEX Ligands for Use in In Vitro Diagnostic, In Vivo Imaging andTherapeutic Delivery.

PhotoSELEX may be used to identify molecules specifically associatedwith a disease condition and/or abnormal cells such as tumor cells.PhotoSELEX-identified oligonucleotides may be produced that reactcovalently with such marker molecules.

In one embodiment of the present invention, the target for photoSELEX isthe abnormal serum or tumor cell (e.g., the target mixture). A librarycandidate mixture of oligonucleotides is generated containingphotoreactive groups. Using one of the above-described photoSELEXprotocols, oligonucleotides able to photocrosslink to the uniqueproteins in the abnormal serum or on the tumor cells are identified.Oligonucleotides able to crosslink to a marker protein on a tumor cellare useful as in vitro diagnostics or when coupled to enhancing agentsfor in vitro imaging. Further, oligonucleotides able to crosslink to amarker protein on a tumor cell may be used therapeutically, for example,as a method for immune activation, as a method of inactivation, or as amethod of delivering specific target-active pharmaceutical compounds.

EXAMPLE 20

PhotoSELEX and HIV-1 Rev.

At each position of the template deoxy-oligonucleotide synthesis, thenucleotide reagent ratio was 62.5:12.5:12.5:12.5. The nucleotide addedin greater amount at each position corresponds to the nucleotide foundin the 6a sequence (SEQ ID NO:5) at the same position.

Cloning and Sequencing procedure: RNA's isolated from each round werereverse transcribed to produce cDNA and PCR amplified producing a 111 bpfragment with unique BamHI and HindIII restriction sites at the ends.The phenol/CHCl₃ treated fragment and a pUC18 vector were digestedtogether overnight with BamHI and HindIII at 37° C., phenol/CHCl₃treated and precipitated. The digested vector and PCR product wasligated at room temperature for 4 hours with T4 DNA ligase andtransformed to competent DH5α-F′ cells which were then grown onampicillin-containing LB plates. Individual colonies were grownovernight in LB-ampicillin media and plasmid was prepared using Wizard(Promega) plasmid preparation kit. Sequencing was performed utilizing aSequenase (USB) kit.

Conditions for nitrocellulose filter binding selections: All roundsutilized approximately 20 nM RNA. Round 1 and 2: 6 nM Rev. Round 3: 3 nMRev. Round 8: 1 nM Rev. Round 9-10: 3 nM Rev. Binding reaction volumesranged from 5 m/s to 1 ml.

Conditions for crosslinking selections: Approximately 50-100 nM offolded pool RNA was added to 0.2 (Rounds 4-6) or 0.5 (Round 7) μM Rev, 1μM BSA in 1×BB (50 mM TrisAc pH 7.7, 200 mM KOAc, 10 mM DTT) on ice andincubated 5 minutes at 370C. The samples were then irradiated at 37° C.,for 3 minutes at 308 nm by a XeCl excimer laser (round 4), 30 minutes at325 nm by a HeCd laser (round 5), 10 minutes at 325 nm, (round 6), or 1minute at 325 nm, (round 7). Approximately one-half of the sample washeated in 50% formamide, 40 μg tRNA at 90° C. for 4 minutes andseparated by electrophoresis in an 8 percent polyacrylamide/8 M ureagel.

The following procedure was utilized to elute crosslinked RNAs fromacrylamide gels with approximately 80% recovery: The nucleoproteincontaining gel slice was crushed to a homogenous slurry in 1×PK buffer(100 mM Tris-Cl pH 7.7,50mM NaCl and 10 mM EDTA). Proteinase K was addedto 1 mg/ml concentration and incubated at 42° C. for 30 minutes. Fifteenminute incubations at 42° C. with increasing urea concentrations ofapproximately 0.7 M, 1.9 M, and 3.3M were performed. The resultingsolution was passed through DMCS treated glass wool and 0.2 μm celluloseacetate filter. The filtered solution was extracted twice withphenol/CHCl₃ and then precipitated with a 1:1 volume mixture ofEtOH:isopropanol.

The crosslinked band from each round was placed in scintillation fluidand counted in a Beckman LS-133 Liquid Scintillation System. The percentcrosslinked=cpms of crosslinked product from RNA +Rev after 4 minutesirradiation at 325 nm minus cpms in crosslinked region for RNA onlyirradiated divided by total cpms. The fold increase in crosslinking is %R13 crosslinked divided by % D37 crosslinked.

Simultaneous selection for affinity and crosslinking using competitortRNA was performed as follows. 10 μM yeast tRNA was added to 0.5 μM Rev,1 μM BSA in 1×BB (50 mM TrisAc (pH=7.5), 200 mM KOAc, 10 mM DTT) andincubated 10 minutes on ice. 200,000 cpms (approximately 50-100 nM finalconcentration RNA) was added and incubated an additional 15 to 60minutes on ice followed by 5 minutes at 37° C. The samples were thenirradiated 4 minutes at 325 nm by a HeCd laser at 37° C. Approximatelyone-third of the sample was heated in 50% formamide, 40 μg tRNA at 90°C. for 4 minutes and separated by electrophoresis in an 8 percentpolyacrylamide-8M urea gel.

The LI crosslinking RNA ligands form additional crosslinked product witha 4 minute 325 nm laser irradiation.

The template oligos used to produce the truncated RNA's are: PTS-1;5′-TAATACGACTCACTATA-3′, (SEQ ID NO:60) DNA-2;5′-GAGTGGAAACACACGTGGTGTCATACACCCTATAGTGAGTCGTATTA-3′ (SEQ ID NO: 61),and DNA-24; 5′-AGGGTTAACAGGTGTGCCTGTTAATCCCCTATAGT-GAGTCGTATTA-3′ (SEQID NO:62). PTS-1 was annealed with DNA-2 or DNA-24 to produce a templatefor T7 transcription.

To calculate the number of changes for individual molecules compared to6a (SEQ ID NO:5), each was aligned to 6a for maximum similarity. Gapsare calculated as one change and truncated molecules were counted asunchanged. To calculate the average probability of finding moleculeswithin each class; the average number of specific (s) and non-specific(ns) changes and unchanged (u) were calculated and used in the equation:

(P)=(0.125)^(s)(0.375)^(ns)(0.625)^(u). Class Ia (P)=9×10⁻¹⁵; Ib

(P)=3×10⁻¹⁵; Ic (P)=7×10⁻¹³; Id (P)=3×10⁻¹⁵; Class II(P)=2×10⁻¹⁴. Sincethe starting population consists of 10¹⁴ molecules, sequences with(P)<10¹⁴ will not be represented. (s) are those changes required toproduce the uppercase, consensus nucleotides and (ns) are additionalchanges.

Trunc24 (SEQ ID NO:59) photo-independent crosslinking with HIV-1 Rev inthe presence of human nuclear extracts was determined as follows:Trunc24 RNA, nuclear extracts, and Rev protein were combined andincubated on ice for 10 min. Samples were mixed 1:1 with 8 M urealoading buffer and placed on a 7 M urea, 8% polyacrylamide gel foranalysis, XL indicates the nucleoprotein complex, RNA indicates freetrunc24 RNA.

EXAMPLE 21

Primer Extension Inhibition Solution SELEX.

Primer extension inhibition relies on the ability of a tightly boundtarget molecule to inhibit cDNA synthesis of high affinityoligonucleotides and results in formation of an amplifiable cDNA poolcorresponding to high affinity oligonucleotides and a non-amplifiableCDNA pool corresponding to low affinity oligonucleotides. Thus, the PCRstep of solution SELEX acts as a partitioning screen between two cDNApools. General protocols for nucleic acid synthesis, primer extensioninhibition and PCR are herein provided. Further, N-acryloylamino phenylmercuric gel electrophoretic conditions for separation of selectednucleic acid ligands is described. The methods of cloning and sequencingnucleic acid ligands is as described by Tuerk and Gold (1990) supra.

RNA Synthesis.

The RNA candidate mixture was generated by incubating RNA polymerase andDNA templates. The reaction conditions are 8% polyethylene glycol 8000,5 mM dithiothreitol, 40 mM Tris-HCl (pH 8.0), 12 mM MgCl₂, 1 mMspermidine, 0.002% Triton X-100, 2 mM nucleotide triphosphates, and 1unit/μl RNA polymerase. Reactions are incubated at 37° C. for 2 hours.

The transcription protocol may be used to generate RNAs with modifiednucleotides. The transcription reaction may either be primed with anucleotide triphosphate derivative (to generate a modified 5′ end),modified nucleotides may be randomly incorporated into the nascent RNAchain, or oligonucleotides or their derivatives ligated onto the 5′ or3′ ends of the RNA product.

Primer Extension Inhibition.

Primer extension inhibition is performed as described by Hartz et al.(1988) supra. Briefly, an oligonucleotide primer is annealed to the 3′end of the oligonucleotides of the candidate mixture by incubating themwith a 2-fold molar excess of primer at 65° C. for 3 min in distilledwater. The annealing reaction is cooled on ice, followed by the additionof {fraction (1/10)} volume of 10×concentrated extension buffer (e.g.,10 mM Tris-HCl (pH 7.4), 60 mM NH₄Cl, 10 mM Mg-acetate, 6 mMβ-mercaptoethanol, and 0.4 mM nucleotide triphosphates). Primerextension is initiated by addition of polymerase and incubation at anyof a variety of temperatures ranging between 0-80° C., and for timesranging from a few seconds to several hours. In one embodiment of themethod of the present invention, primer extension is first conducted inthe presence of chain terminating nucleotide triphosphates such thatlow-affinity nucleic acids preferentially incorporate these chainterminators. A second primer extension is then conducted after removingthe target from high affinity nucleic acids and removing the chainterminating nucleotides triphosphates.

Polymerase Chain Reaction.

The polymerase chain reaction (PCR) is accomplished by incubating anoligonucleotide template, either single- or double-stranded, with 1unit/μl thermal stable polymerase in buffer (50 mM KCl, 10 mM Tris-HCl(pH 8.6), 2.5 mM MgCl₂, 1.7 mg/ml BSA, 1 mM deoxynucleotidetriphosphates, and 1 μM primers). Standard thermal cycles are 95° C. for30 sec, 55° C. for 30 sec, and 72° C. for 1 min, repeated as necessary.One modification of the PCR protocol generates single-strand DNA byincubating either single- or double-stranded template with a single,elongated primer oligonucleotides and results in an elongated product.PCR preferentially amplifies the oligonucleotides rendered amplifiablein the primer extension steps described above.

(N-Acryloylamino)phenyl Mercuric Gel Electrophoresis.

Polyacrylamide gel electrophoresis using N-acryloylamine phenyl mercury(APM) was performed as described by Igloi (1988) Biochemistry 27:3842.APM was synthesized by mixing 8 ml of acetonitrile to 0.35 g of(p-aminophenyl)mercuric acetate at 0° C., followed by 2 ml of 1.2 MNaHCO₃. A total of 0.2 ml of acryloyl chloride was then added withvigorous stirring and the reaction incubated overnight at 4° C. Thesolid phase was collected by centrifugation and washed with water,dissolved by warming to 50° C. in 8.5 ml of dioxane, followed byfiltration to remove undissolved contaminants. APM crystals were formedupon standing at room temperature and the solid was washed again withwater and dried under vacuum. APM was stored at 4° C. APM-polyacrylamidegels were prepared by addition of a appropriate aliquot of a 1 mg/mlsolution of APM in formamide to a solution containing a given amount ofacrylamide, bis(acrylamide), an urea in 0.1 M Tris-borate/EDTA (pH 8.3).Polymerization was initiated by addition of 0.5 ml of 1% ammoniumpersulfate and 7 μl of TEMED per 10 ml of gel solution.

EXAMPLE 22

Enzymatic or Chemical Degradation Solution SELEX.

Enzymes or chemicals may be used to selectively degrade the pool of CDNAcorresponding to low-affinity oligonucleotides. In one embodiment of thepresent invention, restriction enzymes are used to selectively degradethe cDNA pool corresponding to low-affinity oligonucleotides. A numberof restriction enzymes have been identified that cleave single-strandedDNA. These enzymes cleave at specific sequences but with varyingefficiencies.

Restriction enzyme digestion may be performed with a variety of sequencespecific restriction endonucleases. Endonucleases that cleavesingle-stranded DNA include DdeI, HaeIII, HgaI, HinfI, HinPI, MnII,PstI, and RsaI. These enzymes are used under standard conditions knownto those skilled in th -field of molecular biology. Double-strandednucleic acids may also be cleaved using the proper combination ofnucleic acid restriction sequences and site specific restrictionnucleases.

The basic solution SELEX procedure is followed as described in the SELEXPatent Applications. The first cDNA extension is performed in thepresence of four dNTPS, followed by removal of the target. The secondcDNA extension is performed with modified nucleotides that are resistantto enzymatic cleavage by restriction endonucleases. The mixture of cDNAextension products is incubated with the appropriate restriction enzyme.The product of the first cDNA extension from free nucleic acid iscleaved to remove the primer annealing site, rendering this cDNA poolnon-amplifiable by PCR. The efficiency of cleavage by restrictionendonucleases may be improved using a hairpin at the restriction site(RS) to create a localized double-stranded region, as shown in FIG. 24.

Alternatively, the first cDNA extension product is rendered selectivelydegradable by other classes of enzymes by incorporation of modifiednucleotides. For example, cDNA corresponding to low affinity ligands maybe synthesized with nucleotides sensitive to uracil DNA glycosylase,while CDNA corresponding to high affinity ligands may incorporateresistant nucleotides.

Chemical degradation of CDNA corresponding to low affinity ligands canbe accomplished by incorporation of 7-methylguanosine, 5-bromouracil, or5-iodouracil as described using piperidine or photodegradation(Sasse-Dwight and Gralla (1991) Methods Enzymol. 208:146; Aiken andGumport (1991) Methods Enzymol. 208:433; Hockensmith et al. (1991)Methods Enzymol. 208:211).

EXAMPLE 23 Solution SELEX Followed by Affinity Chromatography.

Selective removal of either the first or second cDNA extension productsmay be achieved through affinity chromatography. Removal of the firstcDNA extension product preferentially removes the cDNA poolcorresponding to free or low-affinity nucleic acids. Removal of thesecond cDNA extension product preferentially retains cDNA correspondingto the high-affinity ligand. This strategy relies on the incorporationof modified nucleotides during cDNA synthesis.

Selective Removal of First Extension Product.

Following the basic solution SELEX protocol, the first cDNA extension isperformed in the presence of modified nucleotides (e.g., biotinylated,iodinated, thiolabelled, or any other modified nucleotide) that allowretention of the first cDNA pool on an affinity matrix (FIG. 25). Thetarget is then removed and the second cDNA extension performed in thepresence of non-modified nucleotides. The cDNAs that have incorporatedthe modified nucleotides may be removed by affinity chromatography usinga column containing the corresponding affinity ligand. The CDNA poolcorresponding to nucleic acids with high affinity for the target remainand are then amplified by PCR.

Selective Removal of the Second Extension Product.

Following the basic protocol, the first cDNA extension is performed inthe presence of four dNTPs, and the second cDNA extension is performedin the presence of modified nucleotides (e.g., biotinylated, iodinated,thiolabelled, or any other modified nucleotide) that allow retention ofthe second cDNA pool on an affinity matrix as described above.

Incorporation of Specific Sequences for Annealing to An Affinity Matrix.

In an alternate embodiment of the method of the present invention, aspecial sequence can also be selectively incorporated for annealing toan affinity matrix. Thus, either first or second synthesis cDNAs can beretarded and purified on commercially obtainable matrices as desired.

EXAMPLE 24

Exonuclease Inhibition Solution SELEX.

Exonuclease inhibition may be used to isolate double-stranded ligands.Double-stranded nucleic acid ligands tightly bound to the targetmolecule will inhibit exonuclease hydrolysis at the 3′ edge of thebinding site. This results in a population of nucleic acid moleculesresistant to hydrolysis that also contain a long single-stranded 5′overhang and a central base paired region (see FIG. 26). This nucleicacid molecule is a substrate for any polymerase, and incubation withpolymerase will generate the double-stranded starting material. Thismolecule is amplified by PCR. Members of the nucleic acid candidatemixture that are not tightly bound to the target molecule are digestedduring the initial exonuclease step.

3′→5′ hydrolysis of double-stranded nucleic acid is accomplished byincubation with any double-stranded specific 3′→5′ exonuclease.Exonuclease III specifically hydrolyzes double-stranded DNA 3′→5′ and isactive in a variety of buffers, including 50 mM Tris-HCl (pH 8.0), 5 mMMgCl₂, 10 mM β-mercaptoethanol at 37° C.

EXAMPLE 25

Solution SELEX Method for Isolating Catalytic Nucleic Acids.

Solution SELEX may be used to isolate catalytic nucleic acid sequences.This embodiment of the invention takes advantage of a linear to circulartransformation to sort catalytic nucleic acids from catalytic nucleicacids.

As shown in FIG. 27, the PCR step may be exploited screen the nucleicacid candidate mixture for catalytic members. Catalytic nucleic acidsthat either self-circularize, or alter their 5′ or 3′ ends to allowcircularization with ligase, will amplify during PCR. The figureillustrates circle formation by catalytic members of the candidatemixture; the non-catalytic oligonucleotide members of the candidatemixture will remain linear. After circularization, the candidate mixtureis incubated with a primer that anneals to the extreme 5′ end. In thisembodiment of the invention, only the circular oligonucleotide memberswill generate cDNA and be amplified during the PCR step.

This strategy isolates nucleic acids that either directly catalyzeself-circularization or that modify their own ends so that theamplifiable form may be generated by incubation with ligase. As shown inFIG. 27, the unusual interaction of the cDNA primer with the 5′ end ofthe oligonucleotides of the candidate mixture permits amplification ofonly the circular molecules. In a further embodiment of the method ofthe present invention, this strategy is modified to allow isolation ofcatalytic nucleic acids that catalyze novel reactions.

EXAMPLE 26

Automation of Solution SELEX.

The automated solution SELEX protocol represents a modification of thetechnology used in the automated DNA synthesizer. The nucleic acidcandidate mixture is attached to a solid support by either thebiotin/avidin interaction or a variety of covalent chromatographictechniques (e.g., the condensation of modified nucleotides ontomaleimide or citraconic anhydride supports). The bound nucleic acidcandidate mixture provides a good substrate for targeting binding, andthe column allows use of a single reaction vessel for the SELEXprocedure. Primer extension inhibition is used to physically sort lowand high affinity ligands. Low affinity nucleic acids may be degraded byincorporation of modified nucleotides during the first cDNA extensionstep that renders the cDNA degradable as described in Example 22, whilehigh affinity ligands are copied into non-degradable cDNA and amplifiedby PCR. For additional rounds of solution SELEX, the PCR generatedcandidate mixture is purified or is transcribed into RNA and reattachedto a second solid support, in the same or a new reaction vessel asdesired. The process is repeated as necessary.

64 19 base pairs nucleic acid single linear not provided U at position13 is 5- bromouracil 1 GGGAGCGAGC AAUAGCCGC 19 19 base pairs nucleicacid single linear not provided U at position 13 is 5- iodouracil 2GGGAGCGAGC AAUAGCCGC 19 19 base pairs nucleic acid single linear notprovided U at position 13 has hydrogen molecule attached 3 GGGAGCGAGCAAUAGCCGC 19 44 base pairs nucleic acid single linear not provided all Uare 5-iodouracil 4 GAACAUGAGG AUUACCCAUG AAUUCGAGCU CGCCCGGGCU CUAG 4437 base pairs nucleic acid single linear not provided 5 GGGUGCAUUGAGAAACACGU UUGUGGACUC UGUAUCU 37 36 base pairs nucleic acid singlelinear not provided 6 AGGUACGAUU AACAGACGAC UGUUAACGGC CUACCU 36 37 basepairs nucleic acid single linear not provided 7 UAACGGCUUA ACAAGCACCAUUGUUAACCU AGUGCCU 37 37 base pairs nucleic acid single linear notprovided 8 GAGUGGCUUA ACAAGCACCA UUGUUAACCU AGUACCU 37 36 base pairsnucleic acid single linear not provided 9 GUGCAGAUUA ACAACAACGUUGUUAACUCC UCCUCU 36 37 base pairs nucleic acid single linear notprovided 10 CUGUGGAUUA ACAGGCACAC CUGUUAACCG UGUACCU 37 37 base pairsnucleic acid single linear not provided 11 CUGUGGAUUA ACAGGCACACCUGUUAACCG UGUACCC 37 36 base pairs nucleic acid single linear notprovided 12 AGACGAUUAA CAUCCACGGA UGUUAACGCG CUAGAA 36 37 base pairsnucleic acid single linear not provided 13 AAGACGAUUA ACAAACACGUUUGUUAACGC AACACCU 37 36 base pairs nucleic acid single linear notprovided 14 GAUUGGAUUA ACAGGCACCC CUGUUAACCU ACCACU 36 37 base pairsnucleic acid single linear not provided 15 AGGAGGAUUA ACAACAAAGGUUGUUAACCC CGUACCA 37 34 base pairs nucleic acid single linear notprovided 16 UGAAGGAUUA ACAACUAAUG UUGUUAACCA UGUA 34 37 base pairsnucleic acid single linear not provided 17 UUGAGGAUUA ACAGGCACACCUGCUAACCG UGUACCC 37 37 base pairs nucleic acid single linear notprovided 18 AUGUGGCUUA ACAAGUACGC UUGUUAACCC AAAAACG 37 35 base pairsnucleic acid single linear not provided 19 AGGACGAUGA ACAAACACGUUUGUUCACGC CAUGC 35 38 base pairs nucleic acid single linear notprovided 20 GACUGGCUUA ACAAACAUGU UUUGUUAACC GUGUACCA 38 37 base pairsnucleic acid single linear not provided 21 CGGCGGAUUA ACACGACACACUCGUGUUAA CCAUAUC 37 37 base pairs nucleic acid single linear notprovided 22 GCAUCAGAUG AACAGCACGU CUGUUCACUA UGCACCC 37 37 base pairsnucleic acid single linear not provided 23 GCAUCAGAUG AACAGCACGUCUGUUCACUA UGCACCU 37 37 base pairs nucleic acid single linear notprovided 24 GCAUCAGAUG GACAGCACGU CUGUUCACUA UGCACCU 37 37 base pairsnucleic acid single linear not provided 25 CAGUGUAUGA AACACCACGUGUGUUUCCAC UGUACCU 37 35 base pairs nucleic acid single linear notprovided 26 CAGUGUAUGA AACAACACGU UUGUUUCCAC UGCCU 35 35 base pairsnucleic acid single linear not provided 27 GAGUGUAUGA AACAACACGUUUGUUUCCAC UCCCU 35 35 base pairs nucleic acid single linear notprovided 28 GAGUGUAUGA AACAACACGU UUGUUUCCAC UGUCU 35 35 base pairsnucleic acid single linear not provided 29 GAUUGUAUGA AACAACGUGUUUGUUUCCAC UCCCU 35 35 base pairs nucleic acid single linear notprovided 30 GAAUGUAUGA AACAACACGU UUGUUUCCAC UGCCU 35 37 base pairsnucleic acid single linear not provided 31 GAUUGGACUU AACAGACACCCCUGUUAACC UACCACU 37 34 base pairs nucleic acid single linear notprovided 32 UGCGACAGUU AGAAACACGA UUGUUUACUG UAUG 34 36 base pairsnucleic acid single linear not provided 33 UACAGGCUUA AGAAACACGUUUGUUAACCA ACCCCU 36 36 base pairs nucleic acid single linear notprovided 34 UCGAGCAGUG UGAAACACGA UUGUGUUUCC UGCUCA 36 36 base pairsnucleic acid single linear not provided 35 UGAUGCCUAG AGAAACACAUUAGUGUUUCC CUCUGU 36 37 base pairs nucleic acid single linear notprovided 36 ACGUGCCUCU AGAAACACAU CUGAUGUUUC CCUCUCA 37 37 base pairsnucleic acid single linear not provided 37 ACCCGCCUCG UGAAACACGCUUGAUGUUUC CCUCUCA 37 34 base pairs nucleic acid single linear notprovided 38 CGGUGACGUA UGAAACACGU UCGUUGAUUU CCGU 34 30 base pairsnucleic acid single linear not provided 39 GCUUGCGAAA CACGUUUGACGUGUUUCCCU 30 33 base pairs nucleic acid single linear not provided 40GCACCCUAGA AACGCGUUAG UAGACGUUUC CCU 33 37 base pairs nucleic acidsingle linear not provided 41 AGGAACCUAG AAACACACAG UGUUUCCCUC UGCCCAC37 37 base pairs nucleic acid single linear not provided 42 GCCUGCAUGGAUUAACACGU AUGUGUUAAC CGACUCC 37 37 base pairs nucleic acid singlelinear not provided 43 UGAAACACUG AGAAACACGU GUUUCCCCUU GUGUGAU 37 36base pairs nucleic acid single linear not provided 44 AGGAACCUCAAGCCGCCCCU AGAACACUCG GCACCU 36 37 base pairs nucleic acid single linearnot provided 45 AGGAACCUCA AGAAAGCCCC UGAAACACUC GAAGCCU 37 37 basepairs nucleic acid single linear not provided 46 AGGAACCUCA AGAAACCCCCUGAAACACUC AUUACCG 37 37 base pairs nucleic acid single linear notprovided 47 AGGAACCUCA AGAAAUCCGA ACGACAACCC UACACCU 37 36 base pairsnucleic acid single linear not provided 48 AGGAACCUCA AGAAACCCCGCCACGGACCC CAACCA 36 37 base pairs nucleic acid single linear notprovided 49 GGGAACCUCA AUAAUCACGC ACGCAUACUC GGCAUCU 37 34 base pairsnucleic acid single linear not provided 50 GGGAACCUCA AGAGACCCGACAGGAUACUC GGAC 34 37 base pairs nucleic acid single linear not provided51 AAGUGGAACC UCAAUCCCGU AAGAAGAUCC UGUACCU 37 37 base pairs nucleicacid single linear not provided 52 AUGUGCAUAG AGAUGUACAU AUGAAACCUCAGUAGAG 37 37 base pairs nucleic acid single linear not provided 53UCAUGCAUAG GCAUAGGCAG AUGGAACCUC AGUAGCC 37 37 base pairs nucleic acidsingle linear not provided 54 AUGUGCAACA AGGCGCACGG AUAAGGAACC UCGAAGU37 37 base pairs nucleic acid single linear not provided 55 GAGUACAGCACGCAACACGU ACGGGGAACC UCAAAGU 37 20 base pairs nucleic acid singlelinear not provided N at positions 1 and 20 indicates 1-2 complementarybase pairs N at position 3 indicates 1 or 3 nucleotides N at positions10 and 12 indicates 1-4 complementary base pairs N at position 11indicates 4 or 5 nucleotides U is iodouracil 56 NGNKDAACAN NNUGUUHMCN 2023 base pairs nucleic acid single linear not provided U is iodouracil 57GGAACCUCAA UUGAUGGCCU UCC 23 32 base pairs nucleic acid single linearnot provided U is iodouracil 58 GGGUGUAUGA AACACCACGU GUGUUUCCAC UC 3229 base pairs nucleic acid single linear not provided 59 GGGGAUUAACAGGCACACCU GUUAACCCU 29 17 base pairs nucleic acid single linear notprovided 60 TAATACGACT CACTATA 17 49 base pairs nucleic acid singlelinear not provided 61 GAGTGGAAAC ACACGTGGTG TTTCATACAC CCTATAGTGAGTCGTATTA 49 46 base pairs nucleic acid single linear not provided 62AGGGTTAACA GGTGTGCCTG TTAATCCCCT ATAGTGAGTC GTATTA 46 13 base pairsnucleic acid single linear not provided 63 CTAGAGCCCG GGC 13 44 basepairs nucleic acid single linear not provided 64 CTAGAGCCCG GGCGAGCTCGAATTCATGGG TAATCCTCAT GTTC 44

What is claimed is:
 1. A method for detecting the presence of a targetmolecule that is associated with a disease state in a biologicalsubstance which may contain said target molecule comprising: a) exposinga biological substance which may contain said target molecule that isassociated with a disease state to a nucleic acid ligand identifiedaccording to the method comprising: i) identifying a nucleic acid ligandthat photocrosslinks to a target molecule from a candidate mixture ofnucleic acids, wherein each member of said candidate mixture contains aphotoreactive group, said method comprising: 1) contacting saidcandidate mixture of nucleic acids with a first biological substancewhich contains a target molecule that is associated with said diseasestate, wherein nucleic acids having an increased affinity to a moleculeof said first biological substance relative to the candidate mixtureform nucleic acid-molecule complexes with said molecule; 2) irradiatingsaid complexes, wherein said nucleic acid and molecule photocrosslink;3) partitioning the photocrosslinked nucleic acid-molecule complexesfrom the remainder of the candidate mixture; and 4) identifying nucleicacid ligands that photocrosslink to said target molecule; ii) contactinga second biological substance which does not contain said targetmolecule that is associated with said disease state with said nucleicacid ligands identified in step 4), wherein the nucleic acids withaffinity to a molecule that is not associated with the disease state inthe second biological substance is removed; and iii) amplifying theremaining nucleic acids with specific affinity to said target moleculethat is associated with a disease state to yield a mixture of nucleicacids enriched for nucleic acids with relatively higher affinity andspecificity for binding to said target molecule that is associated withsaid disease state, whereby a nucleic acid ligand to a target moleculethat is associated with a disease state in a biological substance isidentified; b) irradiating said biological substance containing saidnucleic acid ligand; and c) detecting whether a nucleic acidligand-molecule complex has been formed, whereby said target moleculethat is associated with said disease state is detected.
 2. A method fordetecting the presence of a target molecule that is associated with adisease state in a biological substance which may contain said moleculecomprising: a) exposing a biological substance which may contain saidtarget molecule that is associated with a disease state to a nucleicacid ligand identified according to a method comprising: i) identifyinga nucleic acid ligand that photocrosslinks to a target molecule from acandidate mixture of nucleic acids, said method comprising: 1)contacting said candidate mixture of nucleic acids with a firstbiological substance which contains a target molecule that is associatedwith said disease state, wherein nucleic acids having an increasedaffinity to a molecule of said first biological substance relative tothe candidate mixture form nucleic acid-molecule complexes with saidmolecule; 2) partitioning the complexed increased affinity nucleic acidsfrom the remainder of the candidate mixture; 3) amplifying the increasedaffinity nucleic acids to yield a ligand-enriched mixture of nucleicacids, 4) incorporating photoreactive groups into said amplifiedincreased affinity nucleic acids; 5) irradiating said increased affinitynucleic acids, wherein said nucleic acid-molecule complexesphotocrosslink; 6) partitioning the photocrosslinked nucleicacid-molecule complexes from the remainder of the candidate mixture; and7) identifying nucleic acid ligands that photocrosslink to the molecule;ii) contacting a second biological substance which does not contain saidtarget molecule that is associated with said disease with said nucleicacid ligands identified in step vii), wherein the nucleic acids withaffinity to a molecule that is not associated with said disease isremoved; and iii) amplifying the remaining nucleic acids with specificaffinity to said target molecule that is associated with said diseasestate to yield a mixture of nucleic acids enriched for nucleic acidswith relatively higher affinity and specificity for binding to saidtarget molecule that is associated with said disease state, wherebynucleic acid ligands to a target molecule that is associated with adisease state in a biological substance is identified; b) irradiatingsaid biological substance containing said nucleic acid ligand; c)detecting whether a nucleic acid ligand-molecule complex has beenformed, whereby said target molecule that is associated with saiddisease state is detected.